Reversible natural product glycosyltransferase-catalyzed reactions, compounds and related methods

ABSTRACT

The present invention relates to methods of use of glycosyltransferases and related novel compounds. The invention exploits the reversibility of glycosyltransferases to generate new sugars, unnatural biomolecules and numerous one-pot reactions for generation of new biomolecules having varied backbones such as enediynes, vancomycins, bleomycins, anthracyclines, macrolides, pluramycins, aureolic acids, indolocarbazoles, aminglycosides, glycopeptides, polyenes, coumarins, benzoisochromanequinones, calicheamicins, erythromycin, avermectins, ivermectins, angucyclines, cardiac glycosides, steroids or flavinoids. In preferred embodiments, the invention specifically relates to biosynthesis of anticancer (the enediyne calicheamicin, CLM), anthelmintic agents (the macrolides avermectin, ivermectin and erythromycin) and antibiotic (the glycopeptide vancomycin, VCM) natural product-based drugs developed by reversible, bidirectional glycosyltransferase-catalyzed reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/847,731 filed Aug. 30, 2007, which claims priority to U.S.Provisional Application No. 60/824,018, filed Aug. 30, 2006. Both ofthese applications are hereby incorporated by reference herein.

STATEMENT RELATED TO FEDERAL FUNDING

This invention was made with government support under AI052218,CA084374, GM070637 and CA113297 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to methods of usingglycosyltransferases and related novel compounds. The inventionspecifically relates to biosynthesis of anticancer (the enediynecalicheamicin, CLM), anthelmintic agents (the macrolide avermectin,ivermectin and erythromycin) and antibiotic (the glycopeptidevancomycin, VCM) natural product-based drugs developed by reversible,bidirectional glycosyltransferase catalyzed reactions.

BACKGROUND OF THE INVENTION

Glycosyltransferases (GTs) constitute a superfamily of ubiquitousenzymes that attach carbohydrate moieties to biological molecules⁽¹⁾,and thus, play a role in the biosynthesis of oligosaccharides⁽²⁾,glycosaminoglycans⁽³⁾, glycopeptides⁽⁴⁾, and glycosylatedanticancer/anti-infective agents⁽⁵⁾. These enzymes are generallyperceived as unidirectional catalysts that drive the formation ofglycosidic bonds from nucleotide sugar (NDP-sugar) donors and aglyconacceptors⁽⁶⁾.

In practice, these sugar-containing moieties include anticancer agents(the enediyne calicheamicin, CLM), anthelmintic agents (the macrolideavermectin, ivermectin and erythromycin) and antibiotic agents (theglycopeptide vancomycin, VCM) among other compounds. Typically thesenatural product-based drugs are synthesized by unidirectionalGT-catalyzed reactions. However, based on the broad spectrum applicationof these compounds, a greater diversity and availability ofcombinatorial library of these compounds is desirable.

GTs are likely involved in the biosynthesis of anticancer (the enediynecalicheamicin, CLM), anthelmintic (the macrolide avermectin, AVR,ivermectin, and erythromycin) and antibiotic (the glycopeptidevancomycin, VCM) natural product-based drugs which catalyze reversible,bidirectional reactions.

Therefore, a need exists for mechanisms for introducing novel sugarmoieties and conjugating these moieties with varied aglycons to generatebiocombinatorial libraries of these compounds.

SUMMARY OF THE INVENTION

The present invention relates to methods of using glycosyltransferasesand related novel compounds. Generally, the invention exploits thereversibility of glycosyltransferases to generate new sugars, unnaturalbiomolecules and numerous one-pot reactions to generate new biomoleculeshaving varied backbones such as enediynes, vancomycins, bleomycins,anthracyclines, macrolides, pluramycins, aureolic acids,indolocarbazoles, aminglycosides, glycopeptides, polyenes, coumarins,benzoisochromanequinones, calicheamicins, erythromycins, avermectins,ivermectins, angucyclines, cardiac glycosides, steroids or flavinoids.

In one embodiment, the invention specifically relates to biosynthesis ofanticancer agents (the enediyne calicheamicin, CLM), anthelmintic agents(the macrolide avermectin, ivermectin and erythromycin) and antibiotic(the glycopeptide vancomycin, VCM) natural product-based drugs developedby reversible, bidirectional, glycosyltransferase catalyzed reactions.

One exemplary embodiment of the present invention provides a method ofsynthesizing an independent sugar moiety A, in-situ, from a biomoleculehaving a sugar moiety A. This method comprises the steps of: (a)incubating the biomolecule having the sugar moiety A with a nucleotidediphosphate in the presence of a glycosyltransferase, wherein the sugarmoiety A in the biomolecule is excised from the biomolecule, therebygenerating the independent sugar moiety A and a biomolecule aglycon; and(b) isolating the independent sugar moiety A from step (a), wherein thebiomolecule is an enediyne, a vancomycin, a bleomycin, an anthracycline,a macrolide, a pluramycin, an aureolic acid, an indolocarbazole, anaminglycoside, a glycopeptide, a polyene, a coumarin, abenzoisochromanequinone, a calicheamicin, an erythromycin, anavermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.

In this method, the glycosyltransferase is preferably CalG1, CalG2,CalG3, CalG4, GtfD, GtfE, EryBV or AveBI. The biomolecule having thesugar moiety A is an enediyne, a vancomycin, a calicheamicin, anerythromycin, an avermectin or an ivermectin. Further, the sugar moietyis a NDP sugar and the sugar moiety A is a UDP sugar or a TDP sugar.

In one embodiment, the TDP sugar is selected from TDP-α-D-glucose,TDP-β-L-rham nose, TDP-O-methylrhamnose, TDP-6-azidoglucose,TDP-β-L-vancosamine, TDP-β-L-olendrose and TDP-β-L-mycarose. Asdescribed, this synthesis is reversible, whereby incubating theindependent sugar moiety A and the biomolecule aglycon in the presenceof a glycosyltransferase provides the biomolecule having the sugarmoiety A.

Another exemplary embodiment of the present invention provides a methodof exchanging a sugar moiety, in-situ, between (i) an independent sugarmoiety B and (ii) a biomolecule having a sugar moiety A. This methodcomprises the steps of: (a) incubating the independent sugar moiety Bwith the biomolecule having sugar moiety A in the presence of aglycosyltransferase, wherein the sugar moiety A is excised from thebiomolecule and the sugar moiety B is ligated in its place, therebygenerating the independent sugar moiety A and a biomolecule having sugarB; and (b) isolating the independent sugar moiety A and the biomoleculehaving sugar moiety B from step (a), wherein the biomolecule is anenediyne, a vancomycin, a bleomycin, an anthracycline, a macrolide, apluramycin, an aureolic acid, an indolocarbazole, an aminglycoside, aglycopeptide, a polyene, a coumarin, a benzoisochromanequinone, acalicheamicin, an erythromycin, an avermectin, an ivermectin, anangucycline, a cardiac glycoside, a steroid or a flavinoid.

In this method, the glycosyltransferase is preferably CalG1, CalG2,CalG3, CalG4, GtfD, GtfE, EryBV or AveBI. Further, the biomolecule is anenediyne, a vancomycin, a calicheamicin, an erythromycin, an avermectinor an ivermectin and the sugar moiety A or B is independently selectedfrom:

As described here, the sugar exchange is reversible, whereby incubatingthe independent sugar moiety A and the biomolecule having sugar moiety Bin the presence of a glycosyltransferase results in the independentsugar moiety B and the biomolecule having a sugar moiety A.

Yet another exemplary embodiment of the present invention provides amethod of generating a biomolecule A having a sugar moiety A from abiomolecule B having the sugar moiety A, in situ. This method comprisesthe steps of: (a) incubating the biomolecule A, biomolecule B having thesugar moiety A and a nucleotide diphosphate in the presence of aglycosyltransferase wherein (i) the sugar moiety A of the biomolecule Bis excised from the biomolecule B, thereby generating an independentsugar moiety A and a biomolecule aglycon B; and (ii) the independentsugar moiety A and the biomolecule A are ligated, thereby generating thebiomolecule A having the sugar moiety A; and (b) isolating thebiomolecule A having sugar moiety A from step (a), wherein thebiomolecule is an enediyne, a vancomycin, a bleomycin, an anthracycline,a macrolide, a pluramycin, an aureolic acid, an indolocarbazole, anaminglycoside, a glycopeptide, a polyene, a coumarin, abenzoisochromanequinone, a calicheamicin, an erythromycin, anavermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.

In this method the glycosyltransferase is CalG1, CalG2, CalG3, CalG4,GtfD, GtfE, EryBV or AveBI. The biomolecule A or biomolecule B is anenediyne, a vancomycin, a calicheamicin, an erythromycin, an avermectin,an ivermectin or combinations thereof.

As described here, the method of generating biomolecule A having thesugar moiety A from the biomolecule B having the sugar moiety A isreversible, such that incubating the biomolecule A having the sugarmoiety A and the biomolecule aglycon B in the presence of aglycosyltransferase results in the biomolecule B having the sugar moietyA.

Another exemplary embodiment of the present invention provides a methodof generating a biomolecule A having a sugar moiety A and a biomoleculeB having a sugar moiety B from a biomolecule B having the sugar moiety Aand a biomolecule A having the sugar moiety B. This method comprises thesteps of: (a) incubating the biomolecule A having the sugar moiety B,biomolecule B having the sugar moiety A and a nucleotide diphosphate inthe presence of a glycosyltransferase wherein (i) the sugar moiety A ofthe biomolecule B is excised from the biomolecule B, thereby generatingan independent sugar moiety A and a biomolecule aglycon B; (ii) thesugar moiety B of the biomolecule A is excised from the biomolecule A,thereby generating an independent sugar moiety B and a biomoleculeaglycon A; and (iii) the independent sugar moiety A and the biomoleculeA are ligated, the independent sugar moiety B and the biomolecule B areligated, thereby generating the biomolecule A having the sugar moiety Aand biomolecule B having the sugar moiety B; and (b) isolating thebiomolecule A having the sugar moiety A and the biomolecule B havingfrom the sugar moiety B from step (a)(iii), wherein the biomolecule isan enediyne, a vancomycin, a bleomycin, an anthracycline, a macrolide, apluramycin, an aureolic acid, an indolocarbazole, an aminglycoside, aglycopeptide, a polyene, a coumarin, a benzoisochromanequinone, acalicheamicin, an erythromycin, an avermectin, an ivermectin, anangucycline, a cardiac glycoside, a steroid or a flavinoid.

In this method, the glycosyltransferase is preferably CalG1, CalG2,CalG3, CalG4, GtfD, GtfE, EryBV or AveBI. The biomolecule A orbiomolecule B is an enediyne, a vancomycin, a calicheamicin, anerythromycin, an avermectin, an ivermectin or combinations thereof. Asdescribed, this method of generating the biomolecule A having the sugarmoiety A and the biomolecule B having the sugar moiety B is reversible,such that incubating the biomolecule A having the sugar moiety A and thebiomolecule B having the sugar moiety B in the presence of aglycosyltransferase results in the biomolecule B having the sugar moietyA and the biomolecule A having the sugar moiety B.

In yet another exemplary embodiment, the present invention provides amethod of generating a library of isolated glycosylated biomoleculescomprising transferring a sugar moiety from a first biomolecule backboneto a second biomolecule backbone in the presence of aglycosyltransferase wherein the sugar moiety is transferred from thefirst biomolecule backbone to the second biomolecule backbone therebygenerating a non-naturally occurring glycosylated biomolecule, whereinthe biomolecule backbone is an enediyne, a vancomycin, a bleomycin, ananthracycline, a macrolide, a pluramycin, an aureolic acid, anindolocarbazole, an aminglycoside, a glycopeptide, a polyene, acoumarin, a benzoisochromanequinone, a calicheamicin, an erythromycin,an avermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.

In this method, the first and the second glycosylated biomoleculebackbones are independently selected from an enediyne, a vancomycin, acalicheamicin, an avermectin, an ivermectin, an erythromycin andcombinations thereof. The sugar moiety is selected from:

Another embodiment of the present invention provides a glycoside analogof Formula I through XIV having a non-native sugar moiety, wherein theglycoside analog is selected from:

(b) optionally wherein the glycoside analog of Formula II, III, IV or Vfurther includes a 3′-O-methylrhamnose moiety; (c) wherein Y isindependently selected from CH(CH₃)₂, CH₂(CH₃)₂, CH₂CH₃ or CH₃; (d)wherein X is independently selected from H or OH; and (e) wherein, R isindependently selected from a sugar moiety selected from:

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In vitro CalG1-catalyzed reactions. (A) The CalG1-catalyzedtransfer of unnatural sugars to the acceptor (1). The TDP-sugarscorresponding to glycosides 2c-2j were enzymatically generated aspreviously described, TDP-β-L-rhamnose (for 2a) was prepared viachemical synthesis and TDP-α-D-glucose (for 2b) was obtained from acommercial source. (B) CalG1-catalyzed reverse glycosyltransfer andsugar exchange reactions. In the first step, the terminal3′-O-methylrhamnose unit of 2 (CLM α₃ ¹, one of ten CLMs produced by M.echinospora) was transferred to TDP, yielding (1) andTDP-3-O-methyl-β-L-rhamose (3, see also FIGS. 1C and 1D). The subsequentsugar exchange involved the transfer of unnatural sugars (from exogenousNDP-sugars) to (1) to give compounds 2a-2j. (C) Anion exchange HPLC ofCalG1-catalyzed 3 formation: i) control with 50 μN 2 and 100 μM TDP (seealso FIG. 1D, panel ii); ii) 50 μM 2, 100 μM TDP and CalG1 (see alsoFIG. 1D, panel iii). The new peak at 13 min. was isolated and identifiedas 3 by MS/MS (FIG. 9). (D) RP-HPLC of CalG1-catalyzed reactions: i) 50μM (1), 300 μM TDP-β-L-rhamnose and CalG1; if) reverse glycosyltransfercontrol with 50 μM 2 and 100 μM TDP (see also FIG. 1C, panel i); iii) 50μM 2, 100 μM TDP and CalG1 (see also FIG. 1C, panel ii); iv) 50 μM 2,300 μM TDP-3-deoxy-α-D-glucose and CalG1 (sugar exchange); v) 50 μM (1),300 μM TDP-3-deoxy-α-D-glucose and CalG1. All CalG1 assays wereperformed in a total volume of 100 in Tris-HCl buffer (10 mM, pH 7.5)containing 1 mM of MgCl₂ and 10 μM CalG1 with incubation at 30° C. for3-12 h. HPLC parameters are described in the following sections

FIG. 2. Strategy for the construction of a CLM library byCalG1-catalyzed sugar exchange. The general strategy involved theCalG1-mediated exchange of the natural 3′-O-methylrhamnose (highlightedin red) in CLMs α₃ ^(l) (2), β₁ ^(l) (4), γ₁ ^(l) (5), δ₁ ^(l) (6), DMHNac γ (7), γ₂ ^(l) (8), and Nac ε (9) with sugars supplied via the 10established CalG1 NDP-sugar substrates (FIG. 6A). In addition, fragmentIII (10) was also converted to the rhamnoside and glucoside tocumulatively provide 72 diversely functionalized CLM derivatives. Forthis study, CLMs 2 and 4-6 are natural metabolites while 7-10 arechemically modified CLM derivatives. A typical CalG1 sugar exchangereaction contained 50 μN aglycon (2, 4-10), 300 μM NDP-sugar and 10 μMCalG1 in a total volume of 100 μL in Tris-HCl buffer (10 mM, pH 7.5)containing 1 mM of MgCl₂ at 30° C. for 3 h. HPLC parameters are providedin the following sections; chromatograms for representative reactionsare provided in FIG. 10. The structures of all library members areillustrated in FIG. 11 and conversion rates are provided in FIGS. 12 and14. It should also be noted that CalG4 can excise the aminopentosylunits (highlighted in blue) from 4-6 and 8 for sugar/aglycon exchange.

FIG. 3. VCM GT-catalyzed reverse and aglycon exchange reactions. (A)GtfD-catalyzed aglycon exchange reaction to provide2′-vancosaminyl-6′-azidoglucosyl-VCM (15). The TDP-β-L-vancosamine (12)for this reaction was generated in situ by a GtfD-catalyzed reverseglycosyltransfer and subsequently transferred to the unnatural6-azidoglucose-containing derivative 14 to give compound 15 in 27%conversion (FIG. 17). The reaction was performed in a total volume of100 μL in Tricine-NaOH buffer (75 mM Tricine, pH 9.0, 2.5 mM MgCl₂, 2.5mM TCEP and 1 mg/mL BSA) containing 100 μM 11, 100 μM 14, 1 mM TDP and12 μM GtfD. (B) A two component GT-catalyzed aglycon exchange reactionusing two diverse natural product scaffolds. In this one-pot reaction,TDP-6-azidoglucose (16, provided by GtfE-catalyzed reverseglycosyltransfer from sugar donor 14) served as the NDP-sugar donor forthe CalG1-mediated attachment of 6-azidoglucose to CLM 1, yielding 2f in48% conversion (FIG. 18). A typical reaction contained 100 μN 14, 50 μM1, 100 μM TDP, 10 μM GtfE and 10 μM CalG1 in a total volume of 100 μL inTris-HCl buffer (10 mM, pH 7.5) containing 1 mM of MgCl₂ at 30° C. for 3h. For FIG. 3, detailed assay and HPLC parameters and chromatograms areprovided in the following sections.

FIG. 4. Schematic of glycosyltransferase catalysis. (A) The ‘classical’GT-catalyzed sugar transfer from an NDP-sugar donor to an acceptor toform a glycosidic bond. (B) NDP-sugar synthesis via reverseglycosyltransfer. (C) The GT-catalyzed sugar exchange reaction toexchange native natural product sugar appendages with alternative sugarssupplied as exogenous NDP-sugars. (D) A generalized scheme for anaglycon exchange reaction wherein a sugar is excised from one naturalproduct (as an NDP-sugar) and subsequently attached to a distinctaglycon acceptor. In this reaction, the interchange of aglycons from asingle natural product class is generally accomplished via one GT whilethe interchange of aglycons from different compound classes requiresmultiple GTs.

FIG. 5. SDS-PAGE analysis of the purified CLM GTs. Lane 1, CalG4; lane2, CalG1; lane M, standard protein molecular weight markers. CalG1 andCalG4 were overproduced in E. coli BL21 (DE3) and purified asN-(His)₁₀-tagged proteins as described in the Materials and Methods,with overall yields of 10-15 mg per liter of culture.

FIG. 6. Structures of TDP-sugars tested in this work. The sugar donorsin (A) were CalG1 substrates while those in (B) were not. Highlightedparts (red or blue) indicate the structural differences fromTDP-α-D-glucose (II). CalG1 shows the most relaxed specificity to sugarC3-substitution (e.g. V-IX). CalG1 can tolerate neutral modifications atC′6 (I, Ill, IV, IX, X) but not charged substitutions (XI and XV). Withone exception (X), modifications at C4 (XII, XIV, XVI, XVII) and C2(XIII, XVIII and XXI) were not tolerated by CalG1. The generation ofTDP-sugars (III-XXI) was described in “Materials and Methods”, accordingto literature procedures (37-33).

FIG. 7. The sugar substrate flexibility of CalG1. RP-HPLC analysis ofCalG1-catalyzed reactions in the forward direction using PsAg (1) as anacceptor and a pool of TDP-sugar donors (FIG. 6) revealed 10 CalG1substrates. Percent conversions are given in parentheses. The assayswere carried out in a total volume of 100 μl of buffer (10 mM Tris-HCl,pH7.5, 1 mM MgCl₂) containing 50 μN aglycon, 300 μM TDP-sugar, 10 μMCalG1, and were incubated at 30° C. for 12 hrs. Products were confirmedby LC-MS: 1, calc. 1050.1, [M+H] 1051.1; 2a, calc. 1196.2, [M+H]⁺1197.2; 2b, calc. 1212.1, [M+H]⁺ 1213.2; 2c, calc. 1196.2, [M+H]⁺1197.2; 2d, calc. 1196.2, [M+H]⁺ 1197.2; 2e, calc. 1211.2, [M+H]⁺1212.1; 2f, calc. 1237.2, [M+H]⁺ 1238.2; 2g, calc. 1237.2, [M+H]⁺1238.2; 2h, calc. 1195.2, [M+H]⁺ 1196.3; 2i, calc. 1226.2, [M+H]⁺ 1227.3; 2j, calc. 1195.2, [M+H]⁺ 1196.4.

FIG. 8. MS/MS analysis of the regiospecificity of CalG1-catalyzedreactions. Panels A to D show the MS/MS fragmentation of protonatedcompounds 2 (calicheamicin α₃ ^(l), 1211.13⁺, 16.0 eV), 2b (1213.16⁺,16.0 eV), 2d (1197.17⁺, 16.0 eV), and 5 (calicheamicin γ₁ ^(l),1368.12⁺, 16.0 eV), respectively. In panel D, the abundance of the ionsbetween m/z 200-1300 was magnified by 10 fold. The identities of themajor fragment ions produced by glycosidic cleavages are labeled in thescheme below each MS/MS spectrum.

FIG. 9. TDP-dependent CalG1-catalyzed reverse glycosyltransfer. (A)RP-HPLC analysis of the TDP-dependence of CalG1-catalyzed reactionreversibility. TDP mediated the conversion of 2 to 1 (panels iii andiv), with higher [TDP] (2 mM) resulting in a higher conversion (70%)compared to reactions containing lower [TDP] (0.2 mM, 40%). Other NDPsfailed to promote the reverse reaction (e.g. UDP, panel ii). No reactionwas observed in the absence of CalG1 (panel i) or in the presence of adifferent CLM GT (e.g. CalG4, panel v). These control reactionsdemonstrated that the reverse catalysis was specific for TDP and CalG1.(B) Anion exchange HPLC analysis of TDP-sugar formation required forCalG1-catalyzed reverse glycosyltransfer. TDP-3-O-methyl-β-L-rhamnose(3) was observed in CalG1-catalyzed reverse reactions with CLMderivatives 4 (panel vii), 5 (panel viii) and 6 (panel ix). 3 wasseparable from TDP (panel vi, standard) and was absent in a controlreaction performed in the absence of CalG1 (panel x). Reactions werecarried out by co-incubating 50 μM CLMs (2, 4-6) and 100 μM TDP in thepresence or absence of 10 μM CalG1. (C) Characterization ofTDP-3-O-methyl-β-L-rhamnose (3) by MS/MS spectrometry. 3: calc. 562.1,[M−H] 561.0. The MS/MS fragmentation pattern of 3 exhibits several peaksindicative of a TDP-sugar and is illustrated as an inset.

FIG. 10. Representative CalG1-catalyzed ‘sugar exchange’ reactions.RP-HPLC analysis of CalG1-catalyzed reactions with 7 parent CLMderivatives—α₃ ^(l) (2), β₁ ^(l) (4), γ₁ ^(l) (5), δ₁ ^(l) (6), DMH Nacγ₁ ^(l) (7), γ₂ ^(l) (8) and Nac ε₁ ^(l) (9) with TDP-β-L-rhamnose (A)or TDP-α-D-glucose (B). The “DR” designation stands for“derhamnosylated” (meaning removal of the 3′-O-methylrhamnose unit).Structures of “DR” derivatives are available in FIG. 11. The followingcompounds were confirmed by LC-MS: 4, calc. 1381.3, [M+H]⁺ 1382.3; 4DR,calc. 1221.2, [M+H]⁺ 1222.2; 4a, calc. 1367.3, [M+H]⁺ 1368.3; 4b, calc.1383.3, 5, calc. 1367.3, [M+H]⁺ 1368.3; 5DR, calc. 1207.3, [M+H]⁺1208.3; 5a, calc. 1353.3, [M+H]⁺ 1354.3; 5b, calc. 1369.3, [M+H]⁺1370.3; [M+H]⁺ 1384.3; 6, calc. 1353.3, [M+H]⁺ 1354.3; 6DR, calc.1193.3, [M+H]⁺ 1194.3; 6a, calc. 1339.3, [M+H]⁺ 1340.3; 6b, calc.1355.3, [M+H]⁺ 1356.3; 7, calc. 1477.3, [M+H]⁺ 1478.3, [M+Na]+, 1499.3;7DR, calc. 1317.3, [M+Na]+1340.4; 7a, calc. 1463.4, [M+H]+1464.3; 7b,calc. 1479.3, [M+H]⁺ 1480.3; 8, calc. 1335.3, [M+H]⁺ 1336.3; 8DR, calc.1175.2, [M+H]⁺ 1176.2; 8a, calc. 1321.3, [M+H]⁺ 1322.3; 8b, calc.1337.3, [M+H]+1338.3; 9, calc. 1333.3, [M+H]⁺ 1334.3; 9a, calc. 1319.3,[M+H]⁺ 1320.3; 9b, calc. 1335.3, [M+H]⁺ 1336.3.

FIG. 11. The library of CLM analogs. The “DR” designation stands for“derhamnosylated” (meaning removal of the 3′-O-methylrhamnose unit).

FIG. 12. Efficiency of CalG1-catalyzed ‘sugar exchange’ reactions. Thesugar exchange reactions were carried out by co-incubating 50 μM CLM (2,4-9) with 300 of TDP-sugar (FIG. 6A) in the presence of 10 μM CalG1 at30° C. for 3 hrs. The reactions were analyzed by RP-HPLC as described inthe Materials and Methods. The percent conversion for the resultantsugar-exchanged product was calculated from the corresponding HPLCtraces by dividing the integrated area of glycosylated product by thesum of the integrated area of the product and remaining CLM substrate.The slight decrease in sugar exchange efficiency for glycosides c-j ismost likely due to a higher concentration of TDP in these reactions. TDPis a by-product of the nucleotidylyltransferase (E_(p)) reaction bywhich these NDP-sugars were generated. Higher concentrations of TDPfavor the “substrate” side of the equilibrium and disfavor products,thereby lowering sugar exchange efficiency. The following derivativeswere confirmed by LC-MS: 4f, calc. 1408.3, [M+H]⁺ 1409.3; 5c, calc.1353.3, [M+H]+ 1354.3; 5f, calc. 1394.3, [M+H]⁺ 1395.3; 5j, calc.1352.3, [M+H]⁺ 1353.3; 6f, calc. 1380.2, [M+H]⁺ 1381.3; 8f, calc.1362.3, [M+H]⁺ 1363.3; 7f, calc. 1504.4, [M+H]+ 1505.4; 9f, calc.1360.3, [M+H]⁺ 1361.3.

FIG. 13. A representative CalG1-catalyzed aglycon exchange reaction. (A)Scheme for a representative CalG1-catalyzed aglycon exchange. (B)RP-HPLC analysis of CalG1-mediated transformations. (i) Co-incubation of100 μM 4, 50 μM 1 and 0.1 mM TDP in the presence of 10 μM CalG1 led tothe formation of 4DR and 2. (ii) Co-incubation of 100 μM 4 and 0.1 mMTDP in the presence of 10 μM CalG1 led to the production of 4DR. (iii)Co-incubation of 100 μM 2 and 2 mM TDP in the absence of CalG1 resultedin no reaction. (iv) Co-incubation of 100 μM 4 and 2 mM TDP in theabsence of CalG1 resulted in no reaction. The “DR” designation standsfor “derhamnosylated” (meaning removal of the 3′-O-methylrhamnose unit).Compound distributions are indicated in parentheses. All products in (i)were confirmed by LC-MS to give mass values consistent with thosepreviously determined (FIGS. 7, 8 and 10).

FIG. 14. CalG1-catalyzed reverse glycosyltransfer and sugar exchangereactions on a minimal substrate. (A) Scheme for the transformation of10 to 10DR, 10a and 10d. (B) RP-HPLC analysis of CalG1-catalyzedreactions. (i) Co-incubation of 20 μM 10 and 2 mM TDP with 10 μM CalG1led to the formation of 10DR. (ii) Co-incubation of 20 μM 10, 300 μMTDP-β-L-rhamnose and 0.1 mM TDP with 10 μM CalG1 led to the formation ofproduct 10a and by-product 10DR. (iii) Co-incubation of 20 μM 10, 300 μMTDP-a-D-glucose and 0.1 mM TDP with 10 μM CalG1 led to the formation ofproduct 10b and by-product 10DR. (iv) Co-incubation of 20 μM 10 and 2 mMTDP in the absence of CalG1 resulted in no reaction. The “DR”designation stands for “derhamnosylated” (meaning removal of the3′-O-methylrhamnose unit). Compound distributions are indicated inparentheses. The following derivatives were confirmed by LC-MS: 10,calc. 644.1, [M+H]⁺ 645.1; 10DR, calc. 484.0, [M+H]⁺ 485.0; 10a, calc.630.1, [M+H]⁺ 631.1; 10b, calc. 646.1, [M+H]⁺ 647.1.

FIG. 15. CalG4-catalyzed reverse glycosyltransfer. (A) Scheme forCalG4-catalyzed reverse reactions. (B) RP-HPLC analysis of TDP-dependentreverse CalG4 catalysis. (i) Co-incubation of 2 and TDP with CalG4resulted in no reaction, demonstrating that CalG4 did not remove the3′-O-methyl-rhamnose moiety from 2. Co-incubation of TDP and 4 (ii), 5(iii), or 6 (iv) with CalG4 led to the formation of the same reverseglycosyltransfer product 2, demonstrating that CalG4 was specific forthe aminopentose moiety in CLMs. (v) Co-incubation of TDP and 8 withCalG4 led to the formation of product 8DA. The “DA” designation standsfor “de-aminopentosylated”, meaning the removal of the aminopentoseunit. (vi) Co-incubation of TDP and 7 with CalG4 resulted in noreaction, demonstrating the incompatibility of the N-acetyl group withCalG4. These reactions were performed with 2 mM TDP, 50 μN CLMs, and inthe presence or absence of 10 μM CalG4. Co-incubation of UDP (or ADP,CDP, GDP) and CLMs (2, 4-8) with CalG4 resulted in no reaction. Compounddistributions are indicated in parentheses. The product in (v) wasconfirmed by LC-MS: 8DA, calc. 1178.2, [M+H]⁺ 1179.2. The formation of 2in (ii), (iii) and (iv) was confirmed by LC-MS in all cases: calc.,1210.1, [M+H]⁺ 1211.1.

FIG. 16. CalG4-catalyzed aglycon exchange reactions. (A) Scheme forCalG4-catalyzed aglycon exchange reactions. (B) RP-HPLC analysis ofCalG4-catalyzed aglycon exchange reactions. (i) Co-incubation of 4, 1,and TDP with CalG4 led to the formation of product 4DR and by-product 2.(ii) Co-incubation of 5, 1, and TDP with CalG4 led to the formation ofproduct 5DR and by-product 2. (iii) Co-incubation of 6, 1, and TDP withCalG4 led to the formation of product 6DR and by-product 2. (iv)Co-incubation of 8, 1, and TDP with CalG4 led to the formation ofproducts 5DR and by-product 8DA. These reactions were performed using200 μM TDP, 50 μM CLMs (except for 100 μM 6), in the presence of 10 μMCalG4. The “DR” designation stands for “de-rhamnosylated”, meaning theremoval of the 3′-O-methylrhamnose unit. The “DA” designation stands for“de-aminopentosylated”, meaning the removal of the aminopentose unit.Compound distributions are indicated in parentheses. All compounds wereconfirmed by LC-MS analysis to mass values consistent with thosepreviously determined (FIGS. 10 and 15).

FIG. 17. GtfD-catalyzed aglycon exchange. (A) Scheme for arepresentative GtfD-catalyzed aglycon exchange reaction. (B) RP-HPLCanalysis of GtfD-catalyzed reactions. (i) Co-incubation of 100 μM 11,100 μM 14, and 0.1 mM TDP with 12 μM GtfD led to the formation ofproduct 15 and by-product 13. Percent conversion (10%) was calculatedusing the integration areas of peaks 14 and 15. (ii) Co-incubation of100 μM 11, 100 μM 14, and 1 mM TDP with 12 μM GtfD led to the formationof product 15 and by-product 13. Percent conversion (27%) was calculatedusing the integration areas of peaks 14 and 15, revealing a higherconversion upon increasing [TDP]. (iii) Co-incubation of 100 μM 11 and 1mM TDP with 12 μM GtfD led to the formation of 13. (iv) Co-incubation of100 μM 11 and 1 mM TDP in the absence of GtfD resulted in no reaction.Compound distributions are indicated in parentheses. All products wereconfirmed by LC-MS: 11, calc. 1447.4, [M+H]⁺ 1448.4; 13, calc. 1304.3,[M+H]⁺ 1305.2; 14, calc. 1329.3, [M+H]⁺ 1 330.3; 15, calc. 1472.4,[M+H]⁺ 1473.3.

FIG. 18. Determination of the equilibrium constant (K_(eq)) for thenative GtfE-catalyzed reaction. The K_(eq) was measured in duplicate viaa series of reactions under saturation conditions in which the ratio of[TDP]/[TDP-glucose] was varied from 1 to 6 while the initial ratio of[13]/[17] was fixed at 56/44. The change in [13] after a 6 h incubationat 37° C. was determined by RP-HPLC and plotted against[TDP]/[TDP-glucose]. The value of the abscissa axis that corresponds tothe 0-value intercept of the ordinate axis is the uncorrected K_(eq),which was corrected by multiplying by the initial [13]/[17], as thisratio was not exactly 1 (i.e. K_(eq) was determined using the equationK_(eq)=([TDP]/[TDP-glucose]×[13]/[17])).

FIG. 19. Tandem two-GT-catalyzed aglycon exchange. (A) Scheme for theGtfE/CalG1-catalyzed transformation of 1 to 2f. (B) RP-HPLC analysis ofa tandem two-GT-catalyzed reaction. (i) Co-incubation of 100 μM 14, 50μM 1, and 0.1 mM TDP with 10 μM GtfE and 10 μM CalG1 led to theformation of 2f and by-product 17. The percent conversion is indicatedin parentheses. (ii) Co-incubation of 100 μN 14 and 0.1 mM TDP with 10μM GtfE led to the formation of 17 (25%). (iii) Co-incubation of 100 μM14 and 1 mM TDP in the absence of GtfE resulted in no reaction. Productswere confirmed by LC-MS to give values consistent with those previouslydetermined (FIG. 7 and reference 76).

FIG. 20. RP-HPLC analysis of representative AveBI reactions. Panels A-Edepicted the formation of glycosides of 105a-105e in AveBI reactionswith 105 as an acceptor. Panels F-I represented the attachment of xyloseto aglycons 102, 103, 105 and 108 to form 102c, 103c, 105c and 108c byAveBI, respectively. Conversion rates for each reaction were indicatedin parentheses. Assay and HPLC conditions are available in followingsections.

FIG. 21. (A) Tandem Sugar-Assembly by AveBI-catalyzed aglycon-exchangereaction. (B) A library of AVM analogs constructed via AveBI-catalyzedglycorandomization.

FIG. 22. SDS-PAGE analysis of the overexpression and purification ofAveBI from Streptomyces lividans TK64. Lane 1, purified N-His₆-taggedAveBI protein from S. lividans TK64 pCAM4.10; lane 2, soluble fractionsfrom crude extracts of S. lividans TK64 pCAM4.10 expressingN-His₆-tagged AveBI; 2, soluble fractions from crude extracts of S.lividans TK64 pCAM4.11 expressing native AveBI; lane M, proteinmolecular weight standard markers from Invitrogen (Carlsbad, Calif.).The expressed proteins are marked with arrows and molecular weights areindicated on the right column.

FIG. 23. AveBI-catalyzed aglycon exchange reactions. (A) Scheme for aAveBI-catalyzed aglycon exchange reaction. In this reaction,TDP-olendrose (104) was excised from 101 by AveBI and subsequentlytransferred to 105, to produce 106 and 107 in a stepwise, tandem manner.(B) RP-HPLC analysis of AveBI-catalyzed reverse and aglycon exchangereactions. (i) Co-incubation of 100 μM AVM B1a (101, M+Na 895.2) and 2mM TDP in the presence of 12 μM of AveBI yielded 3 (30%, M+Na 751.0).(ii) Co-incubation of 100 μM AVM B1a (101), 100 μM 105 and 2 mM TDP inthe presence of 12 μM of AveBI yielded 103 (63%) from 101, andsubsequently, TDP-oleandrose produced in situ was transferredconsecutively to 105 to yield 106 (28%) and 7 (7%). (iii) Co-incubationof 100 μM IVM (107) and 2 mM TDP in the presence of 12 μM AveBI yielded106 (13%).

FIG. 24. Structures of TDP-sugars tested in this work. The sugar donorsin (A) were AveBI substrates while those in (B) were not. Highlightedparts (red or blue) indicate the structural differences fromTDP-a-D-glucose (XI).

FIG. 25. The reversibility of EryBV-catalyzed reactions.

FIG. 26. RP-HPLC analysis of the exchange reaction of FIG. 25.

FIG. 27. (A) TDP mediated the AveBI reverse catalysis and (B). TDPmediated the EryBV reverse catalysis.

FIG. 28. CalG2 and CalG3 glycorandomization. (A) synthesis directed byCalG2 and CalG3; (B) The CalG2/G3-catalyzed transfer of unnatural sugars28a-I; (C) RP-HPLC of CalG2/G3-catalyzed reactions.

FIG. 29. Representative polyene antibiotics suitable for manipulatingreversible glycosyltransferase reactions according to the presentinvention.

FIG. 30A-E. Representative biomolecules and reversibleglycosyltransferases acting thereupon. Substrate specificity inindicated for each specific glycosyltransferase. FIG. 30A: NysD (5aglycons, 8 NDP sugars), FIG. 30B: GtfE (31 NDP sugars), FIG. 30C: CalG1(8 aglycons, 10 NDP sugars), FIG. 30D: AVrB (5 aglycons, 10 NDP sugars),and FIG. 30E: EryBV (5 aglycons, 10 NDP sugars).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. in General

This invention is not limited to the particular methodology, protocols,and reagents described, as these may vary. One of ordinary skill in theart may change the methodology, synthetic protocols and reagents asnecessary. Further, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims. Unless defined otherwise, all technical and scientificterms used herein have the same meanings as commonly understood by oneof ordinary skill in the art to which this invention belongs.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a sugar” includes aplurality of such sugars and equivalents thereof known to those skilledin the art, and so forth. As well, the terms “a” (or “an”), “one ormore” and “at least one” can be used interchangeably herein. It is alsoto be noted that the terms “comprising”, “including”, “characterized by”and “having” can be used interchangeably.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, exemplary methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference forthe purpose of describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

II. Exemplary Embodiments of the Present Invention

The present invention relates to methods using glycosyltransferases andrelated novel compounds. Generally, the invention exploits thereversibility of glycosyltransferases to generate new sugars, unnaturalbiomolecules and numerous one-pot reactions for generation of newbiomolecules having varied backbones such as enediynes, vancomycins,bleomycins, anthracyclines, macrolides, pluramycins, aureolic acids,indolocarbazoles, aminglycosides, glycopeptides, polyenes, coumarins,benzoisochromanequinones, calicheamicins, erythromycins, avermectins,ivermectins, angucyclines, cardiac glycosides, steroids or flavinoids.

In exemplary embodiments, the invention specifically relates tobiosynthesis of anticancer (the enediyne calicheamicin, CLM),anthelmintic agents (the macrolide avermectin and ivermectin) andantibiotic (the glycopeptide vancomycin, VCM) natural product-baseddrugs developed by reversible, bidirectional glycosyltransferasecatalyzed reactions.

One exemplary embodiment of the present invention provides a method ofsynthesizing an independent sugar moiety A, in-situ, from a biomoleculehaving a sugar moiety A. This method comprises the steps of: (a)incubating the biomolecule having the sugar moiety A with a nucleotidediphosphate in the presence of a glycosyltransferase wherein the sugarmoiety A in the biomolecule is excised from the biomolecule, therebygenerating the independent sugar moiety A and a biomolecule aglycon; and(b) isolating the independent sugar moiety A from step (a), wherein thebiomolecule is an enediyne, a vancomycin, a bleomycin, an anthracycline,a macrolide, a pluramycin, an aureolic acid, an indolocarbazole, anaminglycoside, a glycopeptide, a polyene, a coumarin, abenzoisochromanequinone, a calicheamicin, an erythromycin, anavermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.

In this method, the glycosyltransferase is preferably CalG1, CalG2,CalG3, CalG4, GtfD, GtfE, EryBV or AveBI. The biomolecule having thesugar moiety A is an enediyne, a vancomycin, a calicheamicin, anerythromycin, an avermectin or an ivermectin. Further, the sugar moietyis a NDP sugar and the sugar moiety A is a UDP sugar or a TDP sugar.

In one embodiment, the TDP sugar is selected from TDP-α-D-glucose,TDP-β-L-rham nose, TDP-O-methylrhamnose, TDP-6-azidoglucose,TDP-β-L-vancosamine, TDP-β-L-olendrose and TDP-β-L-mycarose. Asdescribed, this synthesis is reversible, whereby incubating theindependent sugar moiety A and the biomolecule aglycon in the presenceof a glycosyltransferase provides the biomolecule having the sugarmoiety A.

Another exemplary embodiment of the present invention provides a methodof exchanging a sugar moiety, in-situ, between (i) an independent sugarmoiety B and (ii) a biomolecule having a sugar moiety A. This methodcomprises the steps of: (a) incubating the independent sugar moiety Bwith the biomolecule having sugar moiety A in the presence of aglycosyltransferase, wherein the sugar moiety A is excised from thebiomolecule and the sugar moiety B is ligated in its place, therebygenerating the independent sugar moiety A and a biomolecule having sugarB; and (b) isolating the independent sugar moiety A and the biomoleculehaving sugar moiety B from step (a), wherein the biomolecule is anenediyne, a vancomycin, a bleomycin, an anthracycline, a macrolide, apluramycin, an aureolic acid, an indolocarbazole, an aminglycoside, aglycopeptide, a polyene, a coumarin, a benzoisochromanequinone, acalicheamicin, an erythromycin, an avermectin, an ivermectin, anangucycline, a cardiac glycoside, a steroid or a flavinoid.

In this method, the glycosyltransferase is preferably CalG1, CalG2,CalG3, CalG4, GtfD, GtfE, EryBV or AveBI. Further, the biomolecule is anenediyne, a vancomycin, a calicheamicin, an erythromycin, an avermectinor an ivermectin. Further still, the sugar moiety A or B isindependently selected from:

As described here, the sugar exchange is reversible, whereby incubatingthe independent sugar moiety A and the biomolecule having sugar moiety Bin the presence of a glycosyltransferase results in the independentsugar moiety B and the biomolecule having a sugar moiety A.

Yet another exemplary embodiment of the present invention provides amethod of generating a biomolecule A having a sugar moiety A from abiomolecule B having the sugar moiety A, in situ. This method comprisesthe steps of: (a) incubating the biomolecule A, biomolecule B having thesugar moiety A and a nucleotide diphosphate in the presence of aglycosyltransferase wherein (i) the sugar moiety A of the biomolecule Bis excised from the biomolecule B, thereby generating an independentsugar moiety A and a biomolecule aglycon B; and (ii) the independentsugar moiety A and the biomolecule A are ligated, thereby generating thebiomolecule A having the sugar moiety A; and (b) isolating thebiomolecule A having sugar moiety A from step (a), wherein thebiomolecule is an enediyne, a vancomycin, a bleomycin, an anthracycline,a macrolide, a pluramycin, an aureolic acid, an indolocarbazole, anaminglycoside, a glycopeptide, a polyene, a coumarin, abenzoisochromanequinone, a calicheamicin, an erythromycin, anavermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.

In this method the glycosyltransferase is preferably CalG1, CalG2,CalG3, CalG4, GtfD, GtfE, EryBV or AveBI. Also, the biomolecule A orbiomolecule B is an enediyne, a vancomycin, a calicheamicin, anerythromycin, an avermectin, an ivermectin or combinations thereof.

As described here, the method of generating biomolecule A having thesugar moiety A from the biomolecule B having the sugar moiety A isreversible, such that incubating the biomolecule A having the sugarmoiety A and the biomolecule aglycon B in the presence of aglycosyltransferase results in the biomolecule B having the sugar moietyA.

Another exemplary embodiment of the present invention provides a methodof generating a biomolecule A having a sugar moiety A and a biomoleculeB having a sugar moiety B from a biomolecule B having the sugar moiety Aand a biomolecule A having the sugar moiety B. This method comprises thesteps of: (a) incubating the biomolecule A having the sugar moiety B,biomolecule B having the sugar moiety A and a nucleotide diphosphate inthe presence of a glycosyltransferase wherein (i) the sugar moiety A ofthe biomolecule B is excised from the biomolecule B, thereby generatingan independent sugar moiety A and a biomolecule aglycon B; (ii) thesugar moiety B of the biomolecule A is excised from the biomolecule A,thereby generating an independent sugar moiety B and a biomoleculeaglycon A; and (iii) the independent sugar moiety A and the biomoleculeA are ligated, the independent sugar moiety B and the biomolecule B areligated, thereby generating the biomolecule A having the sugar moiety Aand biomolecule B having the sugar moiety B; and (b) isolating thebiomolecule A having the sugar moiety A and the biomolecule B havingfrom the sugar moiety B from step (a)(iii), wherein the biomolecule isan enediyne, a vancomycin, a bleomycin, an anthracycline, a macrolide, apluramycin, an aureolic acid, an indolocarbazole, an aminglycoside, aglycopeptide, a polyene, a coumarin, a benzoisochromanequinone, acalicheamicin, an erythromycin, an avermectin, an ivermectin, anangucycline, a cardiac glycoside, a steroid or a flavinoid.

In this method, the glycosyltransferase is CalG1, CalG2, CalG3, CalG4,GtfD, GtfE, EryBV or AveBI. The biomolecule A or biomolecule B is anenediyne, a vancomycin, a calicheamicin, an erythromycin, an avermectin,an ivermectin or combinations thereof.

As described this method of generating the biomolecule A having thesugar moiety A and the biomolecule B having the sugar moiety B isreversible, such that incubating the biomolecule A having the sugarmoiety A and the biomolecule B having the sugar moiety B in the presenceof a glycosyltransferase results in the biomolecule B having the sugarmoiety A and the biomolecule A having the sugar moiety B.

In yet another exemplary embodiment, the present invention provides amethod of generating a library of isolated glycosylated biomoleculescomprising transferring a sugar moiety from a first biomolecule backboneto a second biomolecule backbone in the presence of aglycosyltransferase wherein the sugar moiety is transferred from thefirst biomolecule backbone to the second biomolecule backbone therebygenerating a non-naturally occurring glycosylated biomolecule, whereinthe biomolecule backbone is an enediyne, a vancomycin, a bleomycin, ananthracycline, a macrolide, a pluramycin, an aureolic acid, anindolocarbazole, an aminglycoside, a glycopeptide, a polyene, acoumarin, a benzoisochromanequinone, a calicheamicin, an erythromycin,an avermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.

In this method, the first and the second glycosylated biomoleculebackbones are independently selected from: an enediyne, a vancomycin, acalicheamicin, an avermectin, an ivermectin, an erythromycin andcombinations thereof. The sugar moiety is selected from:

Another embodiment of the present invention provides a glycoside analogof Formula I through XIV having a non-native sugar moiety, (a) whereinthe glycoside analog is selected from:

(b) optionally wherein the glycoside analog of Formula II, III, IV or Vfurther includes a 3′-O-methylrhamnose moiety; (c) wherein Y isindependently selected from CH(CH₃)₂, CH₂(CH₃)₂, CH₂CH₃ or CH₃; (d)wherein X is independently selected from H or OH; and (e) wherein, R isindependently selected from a sugar moiety selected from:

Glycosyltransferases (GTs), an essential class of ubiquitous enzymes,are generally perceived as unidirectional catalysts. However, thepresent invention teaches that four Gts from two distinct naturalproduct biosynthetic pathways—calicheamicin and vancomycin—catalyzereadily reversible reactions, allowing sugars and aglycons to beexchanged with ease. As proof of the broader applicability of these newreactions, more than seventy differentially glycosylated calicheamicinand vancomycin variants are reported. Thus, the reversibility ofGT-catalyzed reactions may be general and useful for exotic nucleotidesugar generation, establishing in vitro GT activity in complex systems,and enhancing natural product diversity.

Provided below are certain exemplary examples of the preferredembodiment of the invention. These examples are provided forillustrative purposes only and should not be deemed to limit the scopeof the invention.

Example I Exploiting the Reversibility of Natural ProductGlycosyltransferase-Catalyzed Reactions

Specifically, the GTs tested (CLM CalG1/G2/G3/G4 and VCM GtfD/E) werefound to catalyze three new reactions—i) the synthesis of exoticNDP-sugars from glycosylated natural products, ii) the exchange ofnative natural product glycosides with exogenous carbohydrates suppliedas NDP-sugars, and iii) the transfer of a sugar from one natural productbackbone to a distinct natural product scaffold. As proof of the broaderapplicability of these new reactions, the GT-catalyzed production ofmore than seventy differentially glycosylated CLM variants and a VCManalog bearing a handle for chemical diversification and a rare aminosugar are also reported.

The calG1 gene was amplified from the genomic DNA of the CLM-producer,Micromonospora echinospora, overexpressed in E. coli, and therecombinant CalG1 was purified to homogeneity (FIG. 5)⁽⁷⁾. Analysis ofthe CLM γ₁ ^(l) biosynthetic gene cluster revealed four putativeGT-encoded genes, calG1, calG2, calG3 and calG4, implicating a distinctGT for each sugar attachment⁽⁷⁾. The calG1-G4 genes were expressed andpurified to near homogeneity (FIG. 5), with overall yields of 10-15 mgper liter of culture

Incubation of the aglycon (1) with the surrogate substrateTDP-β-L-rhamnose (FIG. 1A) in the presence of CalG1 led to the formationof a new product (FIG. 1D, panel i), characterized as product (2a) byLC-MS.

Consistent with CalG1 as the requisite rhamnosyltransferase in CLMbiosynthesis, no product was observed when CalG1 was replaced with otherGTs in this assay. Also, substitution of TDP-a-L-rhamnose forTDP-β-L-rhamnose in the CalG1 assay yielded no product, consistent withCalG1 functioning as a stereospecific inverting GT.

A diverse library of twenty-two TDP-sugars (Materials and Methods) wasused to probe the NDP-sugar specificity of CalG1 (FIG. 1A and FIG. 6).Nine additional TDP-sugar substrates were converted to theircorresponding CLM glycosides 2b-2j (FIG. 1A) in percent conversions of27%-62% (FIG. 7). LC-MS/MS of products 2b and 2d revealed fragmentationpatterns consistent with attachment of the sugar to the aromatic ring ofthe substrate and were highly consistent with the fragmentation ofnaturally occurring standard CLMs α₃ ^(l) (2) and γ₁ ^(l) (5) (FIGS. 2and 8). Cumulatively, these studies designated CalG1 as the CLMrhamnosyltransferase, capable of flexibility toward diverse TDP-D- andTDP-L-sugar donors.

In an experiment designed to further verify the regiospecificity ofCalG1, CLM α₃ ^(l) (2, FIG. 1B) and TDP-3-deoxy-glucose wereco-incubated with CalG1 under standard conditions. Since the CalG1glycosylation site in (2) is occupied by 3′-O-methylrhamnose (FIG. 1B,3), no reaction was expected. However, two new products were observed,subsequently identified by LC-MS as (1) and the corresponding3-deoxyglucoside (2c) (FIG. 1D, panels iv and v). Analysis of controlreactions led to the conclusion that this transformation involved aTDP-dependent reverse glycosyltransfer. Specifically, co-incubation of(2) with TDP yielded (1) only in the presence of CalG1 (FIG. 1D, panelsii and iii; FIG. 9A) and analysis of the same ‘reverse’ reaction byanion exchange HPLC (FIG. 1C) unveiled the production ofTDP-3-O-methyl-β-L-rhamnose (3, FIG. 1B, FIG. 9) in substantialquantity, which was absent in the control assay. Thus, CalG1 efficientlyexcised the native CLM 3′-O-methylrhamnosyl unit in the presence of TDP(to provide (1) and TDP-sugar (3)) and, in the presence of a slightexcess of exogenous TDP-3-deoxyglucose, ultimately catalyzed theformation of (2c). Such CalG1-catalyzed in situ ‘sugar exchange’ mightoffer an expeditious method for substituting the CLM 3′-O-methylrhamnosewith other natural or unnatural sugars. To test this idea, CLMderivatives (FIG. 2) α₃ ^(l) (2), β₁ ^(l) (4), γ₁ ^(l) (5), δ₁ ^(l) (6),DMH Nac γ (7), γ₂ ^(l) (8), Nac ε (9) and “fragment III” (10)⁽⁹⁾ wereassayed in CalG1-catalyzed reactions with the ten established CalG1TDP-sugar substrates. In every case, the desired sugar-exchanged productwas observed by HPLC (FIGS. 10 and 14) with an average sugar exchangeconversion of 60% for the eight CLM aglycons in the presence of purifiedTDP-α-D-glucose or TDP-β-L-rhamnose. Notably, these simple assays led tothe CalG1-catalyzed production of a CLM library exceeding 70 members(2a-2j, 4a-4j, 5a-5j, 6a-6j, 7a-7j, 8a-8j, 9a-9j, 10a and 10b, FIGS. 11and 12), and thereby highlights the combinatorial power of GT-catalyzed‘sugar exchange’.

Given that GT-catalyzed ‘sugar exchange’ activity proceeds viaestablished NDP-sugar intermediates, GTs may also be used to harvest anexotic sugar from one natural product scaffold and transfer it to adifferent aglycon in a single reaction. This permutation of GT catalysisavoids the often complex synthesis of highly functionalizedNDP-sugars⁽¹⁰⁾. The assays contained CalG1, a putative3′-O-methylrhamnose donor—4, 5, 6, 7, 8 or 10 (FIG. 2)—TDP, and therepresentative acceptor (1).

In each case, the simultaneous excision and in situ transfer of3′-O-methylrhamnose from four to eight or ten to one was observed,yielding the expected 3′-O-methylrhamnosylated product (2) (FIG. 13). Incomparison, controls lacking either CalG1 or TDP gave only startingmaterials. Thus, in situ ‘aglycon exchange’ reactions can extend thepotential diversity accessible by CalG1.

The reversibility of the CalG1-catalyzed ‘sugar exchange’ and ‘aglyconexchange’ transformations described above raised the question as towhether other GT systems would exhibit similar behavior. Thus, threeadditional GT-catalyzed reactions were examined for reversibility—thoseof CalG4 (the putative CLM aminopentosyltransferase), GtfD and GtfE (theVCM vancosaminyl- and glucosyltransferase, respectively)^((7, 11-13)).Christopher T. Walsh (Harvard Medical School, Boston, Mass., USA)provided GtfD and GtfE expression clones. CalG4 was produced in asimilar fashion as CalG1 (FIG. 5)⁽⁷⁾. In the presence of TDP, CalG4catalyzed the excision of the aminopentose sugar moiety (FIG. 2) fromsugar donor CLM derivatives 4, 5, 6, and 8 (FIG. 15). Wyeth Researchprovided some of these CLM analogs.

CalG4 also catalyzed in situ ‘aglycon exchange’, transferring theexcised aminopentoses from donors 4-6 and 8 to the exogenous aglyconacceptor (1) in the presence of TDP with conversions ranging from 19-69%(FIG. 16). In comparison, controls lacking TDP (even in the presence ofalternative NDPs) or CalG4 gave only starting materials. Besidesidentifying CalG4 as the aminopentosyltransferase involved in CLMbiosynthesis, these results confirm that, in contrast to the previouslyproposed UDP-sugar pathways⁽¹⁴⁾, CLM aminopentose biosynthesis proceedsvia a TDP-sugar pathway. Additionally, this demonstrates that thereversibility of GT catalysis is not unique to the CalG1 reaction.

To extend these studies beyond enediyne scaffolds, the VCM GTs GtfD andGtfE were overexpressed and purified as previously described⁽¹¹⁾.Similar to the CLM GTs, GtfD catalyzed the excision of L-vancosaminefrom the parent sugar donor VCM (11) to form pseudoaglycon 13 (FIG. 3A).In a separate aglycon exchange reaction, GtfD catalyzed the transfer ofL-vancosamine from (11) to the unnatural acceptor (14)⁽¹³⁾ to give (15),a VCM analog containing both a sugar-appended azido handle forchemoselective ligation and a vancosaminyl moiety (27% conversion, FIG.3A and FIG. 17). Likewise, the glucosyltransferase GtfE could alsocatalyze sugar excision from both (13) and the unnatural sugar donor(14). Consistent with an equilibrium only moderately favoring theglycoside product in the GtfE-catalyzed reaction, the K_(eq) wasdetermined to be 4.5 (FIG. 18). GtfE could also participate in aglyconexchange, as revealed by the GtfE-catalyzed generation of unnaturalNDP-sugar (16) for CalG1-catalyzed glycosyltransfer to the enediyneacceptor (1) in a tandem, one-pot, GtfE/CalG1-catalyzed aglycon exchangereaction (FIG. 3B). With an overall conversion of 48%, thistransformation highlights the potential of two-GT systems to mediateaglycon exchange between compounds from different natural productclasses (FIG. 19).

The exploitation of GT-catalyzed reaction reversibility may facilitatethe use of glycosylation as a tool to modulate the activity oftherapeutically important natural products⁽⁵⁾. For example, prior tothis work, only two methods for differentially glycosylating CLMs wereavailable—pathway engineering and total synthesis. While the former hasproven to be a powerful derivatization tool for certain naturalproducts⁽¹⁵⁾, the stringent genetic limitations of the CLM-producing M.echinospora has rendered this approach impractical⁽⁷⁾. Alternatively,reworking previously reported CLM syntheses to provide efficientdivergent routes to the >70 CLM analogs reported herein is also likelyimpracticable⁽¹⁷⁻¹⁸⁾. Nicolaou et al. achieved the enantioselectivesynthesis of CLM γ₁ ^(l) in twenty nine steps with an overall yield of0.63%⁽¹⁷⁾ while Danishefsky and coworkers achieved CLM γ₁ ^(l) inseventeen steps with an overall yield of 0.67%⁽¹⁸⁾. With respect to rareNDP-sugars, the demonstrated in situ generation of TDP-β-L-vancosamine(12, FIG. 3A) herein is a significant advance over reported syntheticmethods that required seven linear steps to achieve an overall yield ofless than 7%, originating from the same starting material, VCM⁽¹⁰⁾. TheCLM-derived TDP-3-O-methyl-β-L-rhamnose (3, FIG. 1B) and the threeTDP-N-alkylaminopentoses (derived from donors 4-6 and 8, FIG. 2 and FIG.15) have not been previously synthesized, and therefore, directcomparisons to other synthetic routes are not possible⁽²⁰⁻²²⁾. Advancedsynthetic intermediates related to these NDP-sugars have been reported.As a point of comparison, the simpler substrate TDP-β-L-rhamnose hasbeen prepared by a five-step chemical synthesis with an overall yield of27%⁽²⁰⁾ or a two-step enzymatic method in 62% yield⁽²¹⁾. The mostadvanced intermediate corresponding to the aminopentoses found in theCLMs required eleven linear steps and provided an overall yield of<12%⁽²²⁾.

Although Glaser and Brown described the reversibility of the nativechitin synthetase reaction in one of the first reports of in vitro GTactivity⁽²³⁾, the perception of GT catalysis has remained one ofunidirectionality, transforming NDP-sugar and aglycon substrates intoglycoside products (FIG. 4A)⁽²⁵⁻²⁹⁾.

Cardini et al. first demonstrated the reversibility of the nativesucrose synthetase reaction⁽²⁵⁾, which has subsequently been exploitedto prepare UDP-glucose on large scale⁽²⁶⁾. However, this enzyme isunique among Leloir GTs in that it catalyzes the formation of anunusually high energy sucrose glycosidic linkage (ΔG^(o)−29.3kJ/mol)⁽²⁷⁾. The reversibility of a reaction catalyzed by macrolideresistance GT OleD was implicated by the measurement of its equilibriumconstant (K_(eq)=156)⁽²⁸⁾. Reversibility of the reaction catalyzed bymacrolide GT VinC using a 3-fold molar excess of VinC was also recentlyreported⁽²⁹⁾.

In contrast, this study uncovered reversibility in reactions catalyzedby both previously uncharacterized GTs (CalG1 and CalG4) andwell-studied GTs (GtfD and GtfE) (77-73). Consistent with an equilibriumonly moderately favoring glycoside formation (K_(eq)=4.5 for GtfE),these model GT-catalyzed reactions could be modulated via simpleadjustments in relative substrate concentrations. Glycosyltransferreversibility could be exploited to synthesize valuable rare NDP-sugars(FIG. 4B), exchange one sugar on a core scaffold for another (FIG. 4C),or transfer sugars from one scaffold to another (FIG. 4D), suggesting GTcatalysis to be of significantly greater versatility and utility thanwas previously appreciated.

Materials and Methods

Materials. E. coli DH5α and BL21(DE3) competent cells were purchasedfrom Invitrogen (Carlsbad, Calif.). The pET-16b E. coli expressionvector was purchased from Novagen (Madison, Wis.). Primers werepurchased from Integrated DNA Technology (Coralville, Iowa). Pfu DNApolymerase was purchased from Stratagene (La Jolla, Calif.). Restrictionenzymes and T4 DNA ligase were purchased from New England Biolabs(Ipswich, Mass.). Other chemicals were purchased from Sigma (St. Louis,Mo.). Calicheamicins α₃ ^(l), β₁ ^(l), γ₁ ^(l), γ₂ ¹, DMH Nac γ and Nacε were provided by Wyeth Research (Pearl River, New York).TDP-α-L-rhamnose and TDP-β-L-rhamnose were gifts from Dr. SvetlanaBorisova and Prof. Dr. Hung-wen Liu (University of Texas at Austin,Austin, Tex.). Analytical HPLC was run on a Varian Prostar 210/216system connected to a Prostar 330 photodiode array detector (Varian,Walnut Creek, Calif.). Mass spectra (MS) were obtained by usingelectrospray ionization on Agilent 1100 HPLC-MSD SL quadrupole massspectrometer (Agilent Technologies, Palo Alto, Calif.) connected with aUV/Vis diode array detector.

Chemoenzymatic Synthesis of TDP-sugars. The E_(p) (glucose-1-phosphatethymidyltransferase) reaction was carried out in Tris-HCl buffer (50 mM,pH8.0) containing 5 mM MgCl₂, 1 U inorganic pyrophosphatase, 10 μM ofpurified E_(p), 8 mM sugar-1-phosphate and 6 mM TTP, and incubated at37° C. for 2 h. The formation of TDP-sugars was monitored by RP-HPLC(Phenomenex, Luna C18, 5 μm, 250×4.6 mm, 30 mM KH₂PO₄, pH 6.0, 5 mMtetrabutylammonium hydrogensulfate, 2% CH₃CN with a gradient of 0-50%CH₃CN over 30 min, 1 mL/min, A₂₅₄). The TDP-sugars tested in this studyare highlighted in FIG. 6 and relevant literature citations arepresented in the FIG. 6 legend.

Preparation of CLM 1. A concentrated methanolic solution ofcalicheamicin β₁ ^(l) (4, 15.8 mg, 11.4 μmol) was loaded onto a Dowex50W-X8 (H⁺ form) column (10×1.5 cm) saturated with MeOH, and the columnwas then eluted with 1 L of MeOH. Chromatography was monitored by TLC(CHCl₃/MeOH 10/1 v/v—under these conditions the R_(f) value of (4) is0.2 and (1) is 0.42). The (1)-containing fractions were pooled andevaporated to dryness to give 9.2 mg (8.8 μmol, 77%) final product.

Preparation of 10. Compound 10 was produced by refluxing 10 mg ofcalicheamicin α₃ ^(l) (2) in 10 mL of wet acetone in the presence 0.1equivalents pyridinium p-toluene-sulfonate. Progress of the reaction wasmonitored by RP-HPLC (Phenomenex, Luna C18, 5 μm, 250×4.6 mm, H₂O with a10%-90% CH₃CN gradient over 20 min, 1 mL/min, A280—under theseconditions, calicheamicin α₃ ^(l) eluted at 15.5 min and 10 eluted at11.6 min). After 20 h, acetone was evaporated under pressure and 10 waspurified from the remaining crude reaction mixture by preparativeRP-HPLC (Discovery®BIO C18, 10 μm, 250 mm×21.2 mm, H₂O with a 10%-90%CH₃CN gradient over 20 min, 10 mL/min, A280). Product-containingfractions were pooled and lyophilized to give 0.8 mg (15%) of 10.

Preparation of 13 and 17⁽⁸⁾.

Cloning, expression, and purification of GTs. The calG1 and calG4 genesfrom the calicheamicin producer, Micromonospora echinospora LL6600, wereamplified from genomic DNA by using primer pairs:5′-gccactgaagcttgacttacccatatgctagatatg-3′ (SEQ. ID NO: 1) (forward,NdeI) and 5′-gacggccagatctgagcggtc-3′ (SEQ. ID NO:2) (reverse, BglII)for calC1; 5′-caccggagtgagcatatgcgccagc-3′ (SEQ. ID NO:3) (forward,NdeI) and 5′-gtggacggcagggaatgatcaagatctgggcgcgacc-3′ (SEQ. ID NO:4)(reverse, BglII) for calG4, using Pfu DNA polymerase. PCR products weredigested with NdeI/BglII and ligated into the pET16b expression vector(NdeI/BamHI—to generate the N-terminal MGHHHHHHHHHH fusion) to giveplasmids pCAM2.2 (CalG1) and pCAM10.2 (CalG4), respectively. GtfD andGtfE were expressed according to literature procedures^((16, 19)).

For CalG1 expression, a single transformant of E. coli BL21(DE3)/pCAM2.2 was inoculated into 4 mL LB medium supplemented with 100μg/mL of ampicillin and grown at 37° C. overnight. The precultures wereinoculated into 1 L LB medium with 100 μg/mL of ampicillin and grown at28° C. to an OD₆₀₀ value of 0.5-0.7. Expression was induced with theaddition of 0.4 mM of isopropyl-β-D-thiogalactopyranoside (IPTG)followed by an additional growth for 16 h. The cells obtained from 1 Lof culture were pelleted, washed twice with buffer A (20 mM NaH₂PO₄, pH7.5, 500 M NaCl, 10 M imidazole) and resuspended in 30 mL of buffer Asupplemented with 1 mg/mL of lysozyme. After a 10 min incubation on ice,the proteins were released from the cells by three rounds ofFrench-press (1,200 psi, Thermo IEC), and the insoluble material wasremoved by centrifugation at 30,000×g for 1 hr at 4° C. The supernatantswere loaded onto a HisTrap HT column (1 mL, Amersham Biosciences), andthe N-(His)₁₀-tagged CalG1 was eluted with a linear gradient ofimidazole (10-500 mM) in buffer A via FPLC (Amersham Biosciences). Thepurified protein was desalted through a PD-10 column (AmershamBiosciences) and stored in buffer containing 10 mM Tris-HCl (pH 8.0),100 mM NaCl, and 10% glycerol until use. Protein concentration wasmeasured by Bradford assay⁽²⁴⁾. N-(His)₁₀-tagged CalG4 andN-(His)₆-tagged GtfD, GtfE were purified following the same protocolfrom the appropriate E. coli overexpression strains.

CalG1/CalG4 Assays. CalG1/CalG4 assays were performed in a total volumeof 1004 in Tris-HCl buffer (10 mM, pH 7.5) containing 1 mM of MgCl₂.CalG1-catalyzed reactions in the forward direction were carried out byincubating 50 μM PsAg (1) and 300 μM TDP-sugar (FIG. 6) at 30° C. for3-12 h in the presence or absence of 10 μM CalG1. CalG1-CalG4-catalyzedreactions in the reverse direction were performed by co-incubating 50 μMCLMs (2, 4-10) and 0.2 mM or 2 mM TDP (or UDP, ADP, CDP, GDP) in thepresence of 10 μM CalG1/CalG4. CalG1-catalyzed ‘sugar exchange’reactions were performed by co-incubating 50 μM CLMs (4-10) and 300 μMTDP-sugar (FIG. 6A) in the presence of 10 μM CalG1.CalG1/CalG4-catalyzed ‘aglycon exchange’ reactions were performed byco-incubating 50 μM CLMs (4-10) and 50 μM PsAg (1) in the presence of100 μM TDP and 10 μM CalG1/CalG4. Upon completion, reactions werequenched by the addition of 1004 MeOH, and denatured proteins removed bycentrifugation. The formation of new CLM products was monitored byRP-HPLC (Phenomenex, Luna C18, 5 μm, 250×4.6 mm, 0.1% TFA in H₂O with a10%-100% CH₃CN gradient over 20 min, 1 mL/min, A₂₈₀). Percentconversions were calculated by dividing the integrated area ofglycosylated product by the sum of the integrated area of the productand that of the remaining substrate and products were confirmed by LC-MS(ESI). The formation of TDP-sugars in CalG1-catalyzed reverse reactionswas monitored by anion exchange HPLC (SphereClone SAX, 5 μm, 250×4.60mm, H₂O with 0%-100% 600 mM ammonium formate gradient over 25 min, 1mL/min, A₂₅₄ ⁽³⁰⁾. The peaks corresponding toTDP-3-O-methyl-β-L-rhamnose (3) in CalG1-catalyzed reverse reactionswith (2) were collected evacuated under pressure and lyophilized twiceto remove ammonium formate, prior to ESI-MS analysis.

GtfD/GtfE Assays. Generally, GtfD and GtfE assays were performed in atotal volume of 100 μL Tricine-NaOH buffer (75 mM, pH 9.0) containing2.5 mM MgCl₂, 2.5 M TCEP and 1 mg/mL BSA, as previously reported⁽⁸⁾. TheGtfD-catalyzed reaction in the reverse direction was performed byco-incubating 100 μM vancomycin and 2 mM TDP at 30° C. for 4 h in thepresence of 12 μM GtfD. The GtfD-catalyzed ‘aglycon exchange’ reactionwas performed by co-incubating 100 μM vancomycin (11), 0.1 or 1 mM TDPand 100 μM (14) in the presence of 12 μM GtfD. GtfE-catalyzed reactionsin the reverse direction were carried out by co-incubating 100 μM (14)and 2 mM TDP (or UDP) with 10 μM GtfE at 30° C. for 6 h. Thetwo-GT-catalyzed aglycon exchange reaction was effected by co-incubating100 μM (14), 0.1 mM TDP, 10 μM GtfE, 50 μM (1), and 10 μM CalG1 at 30°C. overnight in Tris-HCl (10 mM, pH7.5) containing 1 mM MgCl₂. Theformation of new vancomycin-analogs was monitored by RP-HPLC using theconditions described previously for the analysis ofCalG1/CalG4-catalyzed aglycon exchange reactions.

Measurement of equilibrium constant (K_(eq)) for the GtfE reaction. Thetime taken for the GtfE-catalyzed native reaction to reach equilibrium(6 h at 37° C.) was first established by measurement of the change inthe concentration of vancomycin pseudoaglycon (13) over time. The K_(eq)for GtfE was measured by fixing the ratio of (13)/(17) at 56/44 (aparallel experiment was fixed at 53/47) and varying the ratios of[TDP]/[TDP-Glc] from 1 to 10. The total concentration of (13)+(17) and[TDP] +[TDP-glucose] was kept at 80 μM and 1 mM, respectively. Thereaction was performed in a total volume of 1004 in Tricine-NaOH buffer(75 mM, pH 9.0) containing 2.5 mM MgCl₂, 2.5 mM TCEP, 1 mg/mL BSA and 10μM GtfE with incubation at 37° C. for 6 h. The change in (13) wasmonitored by RP-HPLC as described previously and plotted against theratio of [TDP]/[TDP-glucose]. The equilibrium constant was subsequentlydetermined from the equation K_(eq)=3)/(17))×([TDP]/[TDP-glucose]).

LC-MS/MS analysis of CLM analogs. LC-ESI-QTOF-MS/MS analysis of the CLManalogs was performed using a capillary LC system (Waters Corp.,Milford, Mass.) coupled to a QTOF Micro mass spectrometer (WatersCorp.). Chromatographic separations were performed on a reverse phasecapillary column (Atlantis® dC18, 3 μm, 75 μm×100 mm). The mobile phasesused were: (A) 5% acetonitrile and 0.1% formic acid in H₂O; (B) 5% H₂Oand 0.1% formic acid in acetonitrile; (C) 0.1% formic acid in H₂O.Samples were loaded onto a trap column (PepMap™ C18, 300 μm×1 mm, 5 μm)using mobile phase C at a flow rate of 30 μL/min for 3 min to desalt thesample. A gradient of mobile phases A and B was then applied (1% Bincrease per min starting at 5% B) at a flow rate of 200 nL/min. Thenanoflow electrospray ionization (ESI) source conditions were set asfollows: capillary voltage 3800V, sample cone voltage 40V, extractioncone voltage 1V, source temperature 120° C., cone gas (N₂) 13 L/hr. TheMS scan was from m/z 100 to 2000. The MS/MS scan was from m/z 50 to 2000at a collision energy of 16 eV.

Referring now to FIG. 28, CalG2 and CalG3 have now been shown to alsoexhibit reversible reactivity and sugar flexibility. The reactionscatalyzed by CalG2 and CalG3 are shown in FIG. 28(A), with productcharacterization by RP-HPLC illustrated in FIG. 28(C). In FIG. 29(B),the inventors demonstrate that CalG2 and CalG3 utilize the various sugarsubstrates shown therein.

Example II Tandem Sugar-Assembly by AveBI-Catalyzed Aglycon ExchangeReaction: Exploitation of a Macrolide Glycosyltransferase for AvermectinGlycorandomization

Avermectins (AVMs, e.g. FIG. 21, 101) are 16-membered macrocycliclactones produced by Streptomyces avermectinius. The avermectins, andthe related C₂₂-C₂₃-reduced ivermectin (e.g. FIG. 21, 107), target theγ-aminobutyric acid (GABA)-related chloride ion channels unique tonematodes, insects, ticks and arachnids, with little or no mammaliantoxicity.³⁴ The widespread commercial use of these remarkableanthelmintic agents began approximately twenty-five years ago asveterinary antiparasitic agents and has more recently expanded toclinical applications for the control of onchocerciasis, stongyloidiasisand lymphatic filariasis. From a biosynthetic perspective, AVMs are oneof only a few known natural products postulated to derive from iterativeglycosylation.³⁵ Specifically, a single glycosyltransferase (GT) isrequired for the attachment of the AVM oleandrosyl-disaccharide (AveBI),proposed to proceed in a stepwise, tandem manner (FIG. 21A).

Evidence in support of iterative glycosylation includes the existence ofa single glycosyltransferase gene (aveBI) within the AVM gene locus,³⁹in vivo studies suggestive of TDP-oleandrose (FIG. 21A, (104)) as animmediate precursor to the AVM oleandrose moiety,³⁷ and the productionof a variety of glycosylated AVMs via in vivo pathway engineering.³⁸

Accordingly, the present invention provides the first definitive invitro biochemical verification of AveBI-catalyzed tandem glycosylation.Furthermore, consistent with the recent illumination of thereversibility of natural product GT-catalyzed reactions,³⁹ theAveBI-catalyzed reaction is shown to also be reversible, the utility ofwhich is demonstrated by generating fifty AVM variants.

The aveBI gene was amplified from pWHM473³⁸ and assessed in severalexpression systems. However, the functional expression of aveBI was onlyachieved in S. lividans TK64 by the use of expression vectors pPWW49 andpPVVW50.⁴⁰ N-His₆-tagged AveBI was subsequently purified to greater than90% purity using HisTrap FPLC purifying system (FIG. 22).

The sequence-confirmed aveBI PCR product was inserted into vector pPWW50to give expression plasmid pCAM4.10, which was introduced intoStreptomyces lividans TK64. The cells expressing N-(His)₆-AveBI wereresuspended in 30 ml of buffer A (20 mM NaH₂PO₄, pH 7.5, 500 mM NaCl, 10mM imidazole) supplemented with 1 mg/ml of lysozyme. The proteins werereleased from the cells by 3 rounds of French-press (1,200 psi). Thesupernatants were loaded onto the HisTrap HT column (1 ml) and theN-(His)₆-tagged AveBI was eluted with a linear gradient of imidazole(10-500 mM) in buffer A by a FPLC system. After desalting through PD-10column the purified AveBI was stored in the buffer containing 25 mMTris-HCl (pH 8.0), 100 mM NaCl and 10% glycerol.

Aglycons 102, 103, 105, 106 and 108 (FIG. 21B) were prepared viaselective acid-mediated hydrolysis of AVM B1a (101) and IVM (7).

Ivermectin (107, 460 mg, 0.525 mmol) was added to a solution of 10 ml 2%H₂SO₄ in isopropanol and stirred at room temperature under argon for 6h. The reaction was stopped via addition of 0.1 ml of triethylamine(NEt₃). Sample was dried, dissolved in 500 μl methanol and loaded onto asilica column (3×30 cm) pre-equilibrated with petroleum. After elutionwith EtOAc/petroleum varying from 0/10, 1/9, 2/8, 3/7, 2/6 (ea. 100 ml),105 (125.9 mg, 0.215 mmol, 41%) and 106 (182.7 mg, 0.250 mmol, 48%) wereobtained with an overall yield of 89%. A small fraction of 108 (1 mg)was also recovered. Similarly, 102 (8.9 mg, 0.015 mmol, 17%) and 103(39.0 mg, 0.053 mmol, 61%) were prepared from AVM B1a (101, 76 mg, 0.087mmol) with an overall yield of 78%. 5. ¹H-NMR (400 Hz, CD₃OD): δ 3.26(d, J=1.8 Hz, 1H), 5.46 (d, J=1.8 Hz, 1H), 1.86 (s, 3H), 4.27 (d, J=5.6Hz, 1H), 3.80 (d, J=5.6 Hz, 1H), 4.63, 4.70 (d, J=14 Hz, 2H), 5.83 (d,J=11.2 Hz, 1H), 5.92 (dd, J=11.2, 14.8 Hz, 1H), 5.72 (dd, 1=14.8, 10 Hz,1H), 2.63 (m, 1H), 1.18 (d, J=6.8 Hz, 3H), 3.99 (br, 1H), 1.57 (s, 3H),5.49 (t, J=8.0 Hz, 1H), 2.32 (t, J=8.0 Hz, 2H), 3.74 (m, 1H), 0.85, 1.95(m, 2H), 5.03 (m, 1H), 2.24 (dd, 1=4.0, 12 Hz, 1H), 1.24 (t, J=12 Hz,1H), 1.5-1.6 (m, 5H), 0.85 (d, J=5.6 Hz, 3H), 3.29 (m, 1H), 1.55 (m,1H), 0.91 (d, J=6.8 Hz, 3H), 1.48 (m, 2H), 1.01 (t, 1=7.4 Hz, 3H);¹³C-NMR (CD₃OD): δ 173.47, 47.10, 120.30, 137.17, 19.93, 69.01, 82.26,81.90, 141.49, 68.71, 121.91, 126.24, 138.38, 41.54, 19.94, 78.43,140.46, 14.96, 118.40, 35.27, 69.14, 37.78, 70.20, 42.98, 98.96, 36.95(2C), 29.39, 18.01, 78.32, 32.59, 12.45, 28.65, 13.00. 106. ¹H-NMR (400Hz, CD₃OD): δ 3.25 (d, J=1.8 Hz, 1H), 5.46 (d, J=1.8 Hz, 1H), 1.85 (s,3H), 4.26 (d, J=5.6 Hz, 1H), 3.80 (d, 1=5.6 Hz, 1H), 4.62, 4.67 (d, J=14Hz, 2H), 5.85 (d, J=11.2 Hz, 1H), 5.91 (dd, J=11.2, 14.8 Hz, 1H), 5.74(dd, J=14.8, 10 Hz, 1H), 2.68 (m, 1H), 1.20 (d, J=7.2 Hz, 3H), 4.02 (br,1H), 1.57 (s, 3H), 5.20 (t, J=7.6 Hz, 1H), 2.33 (t, J=7.6 Hz, 2H), 3.74(m, 1H), 0.83, 1.94 (m, 2H), 5.05 (m, 1H), 2.23 (dd, J=4.0, 12 Hz, 1H),1.27 (m, 1H), 1.5-1.6 (m, 5H), 0.83 (d, J=5.0 Hz, 3H), 3.31 (m, 1H),1.55 (m, 1H), 0.91 (d, J=6.4 Hz, 3H), 1.48 (m, 2H), 0.99 (t, 1=7.4 Hz,3H), 4.83 (d, J=3.2 Hz, 1H), 1.5 (m, 2H), 3.55 (m, J=9.2 Hz, 1H), 3.07(t, J=9.4 Hz, 1H), 3.87 (dd, J=6.2, 9.6 Hz, 1H), 1.25 (d, J=6.2 Hz, 3H),3.48 (s, 3H); ¹³C-NMR (CD₃OD): δ 173.58, 47.09, 120.29, 136.63, 19.94,69.05, 82.27, 82.04, 141.94, 68.74, 121.76, 126.67, 138.39, 41.17,20.98, 83.27, 137.26, 15.39, 120.03, 35.08, 69.03, 37.98, 70.27, 42.95,99.08, 36.94 (2C), 29.44, 17.97, 77.81, 32.60, 12.76, 28.45, 13.01,96.55, 35.98, 79.57, 77.64, 70.00, 18.27, 57.67.

The reversibility of the AveBI reaction was examined usingcommercially-available 101 and TDP.

The chemoenzymatic synthesis of sugar nucleotide (TDP-β-L-olivose)required six linear steps with an overall reported yield of 20%.

RP-HPLC analysis of an in vitro assay containing 50 μM 101, 2 mM TDP and12 μM AveBI revealed the formation of 103 from 101 (30%, FIG. 21A andFIG. 23), while 101 remained unchanged in control assays lacking eitherTDP or AveBI.

Generally, AveBI assays were performed in a total volume of 100 I inTris-HCl buffer (50 mM, pH 8.0) containing 2 mM MgCl₂. Reversibility ofAveBI reaction was assayed by co-incubation of 100 μM avermectin B1a (1)or ivermectin (7) and 2 mM TDP with 12 μM AveBI at 30° C. overnight. TheAveBI-catalyzed aglycon exchange reaction was assayed by co-incubationof 100 M (1), 100 μM (5) and 2 mM TDP with 12 μM AveBI at 30° C.overnight.

To assess whether AveBI was capable of catalyzing an ‘aglycon exchange’reaction,³⁹ a reaction containing 100 μM (01), 100 μM (105), 2 mM TDPand 12 μM AveBI was subsequently analyzed. Examination of this reactionrevealed the production of TDP-oleandrose (104) from (103) (63%) and thesubsequent transfer of oleadrose to (105), to provide (106) (28%) andtrace amounts of (107) (7%) (FIG. 21A, FIG. 23). Cumulatively, thesestudies unequivocally establish AveBI as the GT responsible for thestepwise tandem assembly of the AVM oleandrosyl disaccharide and revealthe AveBI-catalyzed reaction to be readily reversible and amenable to‘aglycon exchange’ transglycosylation strategies.³⁹

The AveBI sugar nucleotide specificity was subsequently probed withtwenty-two NDP-sugars (generated chemically or chemoenzymatically, FIG.24).⁴¹ As a representative example, IVM aglycone (105) withTDP-6-deoxyglucose led to a new product (99% conversion, FIG. 20A), theLC-MS of which was consistent with the anticipated product (105a) (FIG.21B).

The reaction contained 50 μM algycon (101-103, 105-108), approximate 300μM TDP-sugar and 12 μM AveBI, and was incubated at 30° C. overnight.

Substituting TDP-6-deoxyglucose with UDP-6-deoxyglucose in the sameassay gave (105a) in only 10% yield, indicating a preference forTDP-sugars. Further AveBI-IVM assays revealed that nine additionalTDP-sugar substrates were converted to their corresponding IVMglycosides 105b-105j (FIG. 21B). In a similar fashion, the same setsugars were transferred to aglycons 102, 103, 106 and 108, producingglycosides 102a-102j, 103a-103j, 106a-106j and 108a-108j (FIG. 21, FIG.20), respectively. The conversion rates for a-e glycosides ranged from18% to 99% while only trace production (1%-10%) of f-j glycosides wasobserved, except for 106 h (25%) and 106g (19%). All products wereconfirmed by LC-MS (supporting information, Table 1). Consistent withthe previous in vivo studies,³⁸ no tandem addition of D-configuredsugars to aglycon (102) and (105) or trisacchride AVM derivatives wasobserved in this study.

TABLE 1 LC-MS characterization of AVM analogues. Com- Con- Retentionpound version time MS (m/z) No. rate (%) (min) calcd [M + H]⁺ [M + Na]⁺[M − H]⁻ 102 / 16.6 584.3 585.2 587.0 584.3 102a 94.4% 12.3 730.4 731.2729.4 102b 52.9% 17.1 714.4 737.2 713.4 102c 28.9% 10.5 716.4 739.2715.4 102d 13.6% 7.7 729.4 730.4 768.2 728.2 102e 22.0% 7.4 729.4 730.2768.2 729.0 102f  2.5% 13.4 771.4 772.2 794.2 770.4 102g  7.2% 14.2728.4 729.2 751.2 727.4 102h  6.1% 10.2 730.4 753.4 729.4 102i  1.0%10.2 730.4 753.4 729.4 102j  1.0% 13.2 771.4 772.2 794.2 770.4 103 /21.2 728.4 729.0 751.0 727.4 103a 93.1% 18.5 874.5 875.3 897.2 873.4103b 84.7% 25.1 858.5 859.2 881.2 857.5 103c 86.5% 15.9 860.5 861.2883.2 859.5 103d 18.8% 8.7 873.5 874.2 872.4 103e 19.9% 8.2 873.5 874.0896.2 872.5 103f  2.4% 20.3 915.5 916.0 938.0 914.4 103g  6.7% 23.7872.5 873.2 895.0 871.5 103h  8.0% 14.6 874.5 897.2 874.4 103i  5.7%14.6 874.5 897.0 873.2 103j  2.5% 18.3 915.5 938.0 914.4 105 / 22.1586.4 587.2 585.4 105a 98.5% 15.3 732.4 733.2 755.0 731.4 105b 48.7%23.8 716.4 716.8 739.0 715.4 105c 20.3% 13.2 718.4 719.2 717.4 105d20.8% 8.7 731.4 732.0 754.0 730.4 105e 24.4% 8.1 731.4 732.0 754.0 730.4105f  9.6% 21.4 773.4 774.2 796.0 772.4 105g 22.4% 18.8 730.4 731.2729.4 105h  5.1% 12.9 732.4 755.0 731.4 105i  1.6% 14.2 732.4 755.0731.4 105j  1.2% 15.4 773.4 796.6 772.4 106 / 29.5 730.4 753.0 729.4106a 98.5% 26.9 876.5 899.0 875.4 106b 85.2% 34.4 860.5 883.2 859.5 106c73.6% 23.1 862.5 885.0 861.4 106d 18.2% 8.1 875.5 876.2 874.2 106e 30.3%8.0 875.5 876.2 898.2 874.5 106f  5.1% 28.8 917.5 940.2 916.4 106g 18.7%33.1 874.5 897.2 873.5 106h 24.9% 20.6 876.5 899.2 875.4 106i  9.2% 20.7876.5 899.0 875.2 106j  5.4% 25 917.5 940.0 916.4 108 / 26.1 716.4 739.2715.4 108a 95.3% 23.5 862.5 885.2 861.4 108b 90.5% 31.6 846.5 869.2845.2 108c 80.8% 20.2 848.5 871.2 847.4 108d 27.4% 9.5 861.5 862.2 884.2860.4 108e 17.8% 8.8 861.5 862.2 884.2 860.4 108f  5.1% 25.6 903.5 926.2902.4 108g  7.0% 28.4 860.5 861.2 883.2 859.5 108h  4.4% 18.0 862.5885.2 861.5 108i 11.6% 18.1 862.5 885.4 861.4 108j  6.2% 22.2 901.5902.2 924.4 900.4

Materials and Methods

Materials. E. coli DH5a and BL21 (DE3) competent cells were purchasedfrom Invitrogen. The E. coli expression vectors pET-11a, pET28a werepurchased from Novagen. The plasmids, pPWW49 and pPWW50, for expressionin S. lividans, were generous gifts from Dr. Udo F. Wehmeier and Prof.Dr. Wolfgang Piepersberg (Bergische University, Wuppertal, Germany).Primers were ordered from Integrated DNA Technology. Pfu DNA polymerasewas purchased from Stratagene. Restriction enzymes and T4 DNA ligasewere purchased from New England Biolabs. Other chemicals were purchasedfrom Sigma (St. Louis, Mo.). TDP-α-L-rhamnose and TDP-β-L-rhamnose weregifts from Dr. Svetlana Borisova and Prof. Dr. Hung-wen Liu (Universityof Texas at Austin, Austin, USA). Ivermectin was purchased from Sigma(St. Louis, Mo.) and avermectin B1a was purchased from Supelco(Bellefonte, Pa.). ¹H NMR, ¹³C NMR and two-dimensional correlationspectra (gCOSY, TOCXY, gHSQC and gHMBC) were recorded in CD₃OD on a400-MHz Varian INOVA model NMR spectrometer. Chemical shifts arereported in parts per million (ppm, δ) relative to CD₃OD (0.00). ¹H NMRsplitting patterns with observed first-order coupling are designated assinglet (s), doublet (d), or triplet (t). Splitting patterns that couldnot be interpreted or easily visualized are designated as multiplet (m).Mass spectra (MS) were obtained by using electrospray ionization onAgilent 1100 HPLC-MSD SL quadrupole mass spectometer connected with aUV/Vis diode array detector.

Chemoenzymatic Synthesis of TDP-sugars. The E_(p) (glucose-1-phosphatethymidylyltransferase) reaction was carried out in Tris-HCl buffer (50mM, pH8.0) containing 5 mM MgCl₂, 1U inorganic pyrophosphatase, 10 μM ofpurified E_(p), 8 mM sugar-1-phosphate and 6 mM TTP, and incubated at37° C. for 2 h. The formation of TDP-sugars was monitored by RP-HPLC(Phenomenex, Luna C18, 5 μm, 250×4.6 mm, 30 mM KH₂PO₄, pH 6.0, 5 mMtetrabutylammonium hydrogensulfate, 2% CH₃CN with a gradient of 0-50%CH₃CN over 30 min, 1 mL/min, A₂₅₄).

Chemical Preparation of Algycons. Ivermectin (107, 460 mg, 0.525 mmol)was added to a solution of 10 ml 2% H₂SO₄ in isopropanol and stirred atroom temperature under argon for 6 h. The reaction was stopped viaaddition of 0.1 ml of triethylamine (NEt₃).⁴³ Sample was dried,dissolved in 500 μl methanol and loaded onto a silica column (3×30 cm)pre-equilibrated with petroleum. After elution with EtOAc/petroleumvarying from 0/10, 1/9, 2/8, 3/7, 2/6 (ea. 100 ml), 105 (125.9 mg, 0.215mmol, 41%) and 106 (182.7 mg, 0.250 mmol, 48%) were obtained with anoverall yield of 89%. A small fraction of (108) (1 mg) was alsorecovered. Similarly, (102, 8.9 mg, 0.015 mmol, 17%) and (103, 39.0 mg,0.053 mmol, 61%) were prepared from AVM B1a (101, 76 mg, 0.087 mmol)with an overall yield of 78%.

NMR data for (105) and (106). 105. ¹H-NMR (400 Hz, CD₃OD): δ 3.26 (d,J=1.8 Hz, 1H), 5.46 (d, J=1.8 Hz, 1H), 1.86 (s, 3H), 4.27 (d, J=5.6 Hz,1H), 3.80 (d, 1=5.6 Hz, 1H), 4.63, 4.70 (d, J=14 Hz, 2H), 5.83 (d,j=11.2 Hz, 1H), 5.92 (dd, J=11.2, 14.8 Hz, 1H), 5.72 (dd, 1==14.8, 10Hz, 1H), 2.63 (m, 1H), 1.18 (d, J=6.8 Hz, 3H), 3.99 (br, 1H), 1.57 (s,3H), 5.49 (t, J=8.0 Hz, 1H), 2.32 (t, J=8.0 Hz, 2H), 3.74 (m, 1H), 0.85,1.95 (m, 2H), 5.03 (m, 1H), 2.24 (dd, 1=4.0, 12 Hz, 1H), 1.24 (t, J=12Hz, 1H), 1.5-1.6 (m, 5H), 0.85 (d, J=5.6 Hz, 3H), 3.29 (m, 1H), 1.55 (m,1H), 0.91 (d, J=6.8 Hz, 3H), 1.48 (m, 2H), 1.01 (t, 1=7.4 Hz, 3H);¹³C-NMR (CD₃OD): δ 173.47, 47.10, 120.30, 137.17, 19.93, 69.01, 82.26,81.90, 141.49, 68.71, 121.91, 126.24, 138.38, 41.54, 19.94, 78.43,140.46, 14.96, 118.40, 35.27, 69.14, 37.78, 70.20, 42.98, 98.96, 36.95(2C), 29.39, 18.01, 78.32, 32.59, 12.45, 28.65, 13.00.

(106). ¹H-NMR (400 Hz, CD₃OD): δ 3.25 (d, J=10.8 Hz, 1H), 5.46 (d, J=1.8Hz, 1H), 1.85 (s, 3H), 4.26 (d, J=5.6 Hz, 1H), 3.80 (d, J=5.6 Hz, 1H),4.62, 4.67 (d, J=14 Hz, 2H), 5.85 (d, J=11.2 Hz, 1H), 5.91 (dd, J=11.2,14.8 Hz, 1H), 5.74 (dd, 1=14.8, 10 Hz, 1H), 2.68 (m, 1H), 1.20 (d, J=7.2Hz, 3H), 4.02 (br, 1H), 1.57 (s, 3H), 5.20 (t, 1=7.6 Hz, 1H), 2.33 (t,1=7.6 Hz, 2H), 3.74 (m, 1H), 0.83, 1.94 (m, 2H), 5.05 (m, 1H), 2.23 (dd,J=4.0, 12 Hz, 1H), 1.27 (m, 1H), 1.5-1.6 (m, 5H), 0.83 (d, J=5.0 Hz,3H), 3.31 (m, 1H), 1.55 (m, 1H), 0.91 (d, J=6.4 Hz, 3H), 1.48 (m, 2H),0.99 (t, J=7.4 Hz, 3H), 4.83 (d, 1=3.2 Hz, 1H), 1.5 (m, 2H), 3.55 (m,1=9.2 Hz, 1H), 3.07 (t, 1=9.4 Hz, 1H), 3.87 (dd, 1=6.2, 9.6 Hz, 1H),1.25 (d, J=6.2 Hz, 3H), 3.48 (s, 3H); ¹³C-NMR (CD₃OD): δ 173.58, 47.09,120.29, 136.63, 19.94, 69.05, 82.27, 82.04, 141.94, 68.74, 121.76,126.67, 138.39, 41.17, 20.98, 83.27, 137.26, 15.39, 120.03, 35.08,69.03, 37.98, 70.27, 42.95, 99.08, 36.94 (2C), 29.44, 17.97, 77.81,32.60, 12.76, 28.45, 13.01, 96.55, 35.98, 79.57, 77.64, 70.00, 18.27,57.67.

Cloning, expression and purification of AveBI. The aveB/gene wasamplified from pWHM473 using primers5′-ctagacagtgacatatgtcagatcattttctcttc-3′ (SEQ. ID NO:5) (forward, NdeI)and 5′-aaccctgtgagatctactcaccgcccggc-3′ (SEQ. ID NO:6) (reverse, BglII).The PCR products were cut with NdeI/BglII and inserted into pPCPU21(NdeI/BglII), resulted in plasmid pCAM4.9. After confirmation bysequencing, the aveBI insert was cut with NdeI/BgAI from pCAM4.9 andligated to vectors pET11a, pET16b, pPWW49 and pPWW50 (NdeI/BamHI),⁴⁴resulted in expression plasmids pCAM4.1, pCAM4.2, pCAM4.11 and pCAM4.10,respectively. Soluble expression of AveBI was only achieved inStreptomyces lividans TK64 harboring pCAM4.11 or pCAM4.10. Specifically,the plasmid pCAM4.10 was introduced into S. lividans TK64 by standardtransformation,⁴⁵ for the expression of N-(His)₆-AveBI. Thetransformants were grown in liquid TSB media (thiostrepton 25 μg/ml) forthree days at 28° C. and were transferred to YEME media (thiostrepton 25μg/ml) containing 25% sucrose. The culture was incubated at 28° C. fortwo more days and cells were harvested. The pellets obtained from 300 mLof culture were washed twice with buffer A (20 mM NaH₂PO₄, pH 7.5, 500mM NaCl, 10 mM imidazole) and resuspended in 30 ml of buffer Asupplemented with 1 mg/ml of lysozyme. After a 10 min incubation on ice,the proteins were released from the cells by three rounds ofFrench-press (1,200 psi, Thermo IEC) and the insoluble material wasremoved by centrifugation at 30,000 g for 1 hr (4° C.). The supernatantswere loaded onto the HisTrap HT column (1 ml, Amersham Biosciences) andthe N-(His)₆-tagged AveBI was eluted with a linear gradient of imidazole(10-500 mM) in buffer A by a FPLC system (Amersham Biosciences). Thepurified protein was desalted through PD-10 column (AmershamBiosciences) and stored in the buffer containing 25 mM Tris-HCl (pH8.0), 100 mM NaCl and 10% glycerol until use. Protein concentration wasmeasured by Bradford assay.⁴⁶

AveBI assays. Generally, AveBI assays were performed in a total volumeof 100 μl in Tris-HCl buffer (50 mM, pH 8.0) containing 2 mM MgCl₂.Reversibility of AveBI reaction was assayed by co-incubation ofavermectin B1a (101, 100 μM) and TDP (2 mM) with 12 μM AveBI at 30° C.overnight. The AveBI-catalyzed aglycon exchange reaction was assayed byco-incubation of 100 μM (101), 100 μM (105) and 2 mM TDP with 12 μMAveBI at 30° C. overnight. To probe AveBI sugar substrate specificity,the reaction contained 50 μM algycon (101-103, 105-108) and approximate300 μM TDP-sugar (directly from E_(p) reactions) in the presence of 12μM AveBI and was incubated at 30° C. overnight. The reactions wereanalyzed by HPLC using a reversed phase column Luna C18, 5 μm, 250×4.6mm (Phenomenex) with UV detection at 243 nm. The following elutionprofile was used: solvent system (solvent A, 0.1% TFA in water; solventB, acetonitrile), 30% B to 70% B (linear gradient, 0-5 min), 70% B to100% B (linear gradient, 5-25 min); 100% B (25-30 min); 100% B to 30% B(linear gradient, 30-31 min) and 30% B (31-40 min).

In summary, the present invention provides indisputable evidence of theAveBI-catalyzed tandem sugar addition within AVM biosynthesis. Further,the demonstrated promiscuity of AveBI further highlights the inherentflexibility of many secondary metabolite GTs and provides a rapidone-pot strategy for the generation of 50 differentially-glycosylatedAVMs. In contrast to the macrolide in vitro GT studies to date,⁴² AveBIdoes not require a helper protein for activity. Finally, the recentlyestablished ‘sugar/aglycon exchange’ strategies,³⁹ and the concept ofreversibility of GT-catalyzed reactions to provide exotic sugarnucleotides, are shown to apply to macrolides.

Example III Exploiting the Reversibility ofGlycosyltransferase-catalyzed Reactions for CombinatorialDiversification of Macrolides Reversibility of EryBV-Catalyzed Reactions(FIG. 25)

As shown in FIG. 25, TDP mediated the reverse catalysis of EryBV toexcise mycarose from 3-a-mycarosyl erythronolide B (206) to formerythronolide B (208). EryBV also catalyzed the transformation oferythromycin B (202) and erythromycin D (204) into (205), but had noreverse activity on erythromycin A (201) and erythromycin C (203). In anEryBV-catalyzed ‘aglycon exchange’ reaction, TDP-mycarose (207) wasproduced in a reverse catalysis from 206 and was subsequentlytransferred to 6-deoxyerythronolide B (209) to yield a new macrolide(210). The corresponding RP-HPLC analysis of this exchange reaction wasdepicted in FIG. 26.

Combinatorial ‘Aglycon Exchange’ Reactions

As shown in FIG. 27, 27A. TDP mediated the AveBI reverse catalysis toproduce TDP-oleandrose (212) from (211), (212) was subsequentlytransferred by EryBV to (208) (or 209) to yield new macrolides (214) or(215). Further, as shown FIG. 27B, TDP mediated the EryBV reversecatalysis to produce TDP-mycarose (207) from (206), (207) wassubsequently transferred by AveBI to (216) in a stepwise, tandemreaction to produce new avermectin derivatives (217) and (218).

Example IV Exploitation of Glycosyltransferase for PolyeneGlycorandomization

FIG. 29 illustrates five polyene antibiotics which are acted upon byreversible glycosyltransferase NysD1. NysD1, the glycosyltransferasewhich glycosylates Nystatin A1, has been shown to exhibit flexibility inaglycon and sugar donor specificities. NysD1 has been shown capable ofacting upon five different polyene aglycons and eight differentNDP-sugars.

The present invention exploits the reversibility of glycosyltransferasesto generate new, unnatural biomolecules. The broad utility of thisinvention is seen in FIGS. 30A-E, where five different scaffolds areshown, upon which various glycosyltransferases can act, and upon whichreversible GTs act, each GT being capable of utilizing multiple aglyconsand sugar donors.

Those skilled in the art will recognize, or be able to ascertain usingno more then routine experimentation, numerous equivalents to thespecific compounds, protocols, methods, assays and reagents describedherein. Such equivalents are considered to be within the scope of thisinvention and covered by the following claims. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

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What is claimed is:
 1. A method of exchanging a sugar moiety, in-situ,between (i) an independent sugar moiety B and (ii) a biomolecule havinga sugar moiety A, the method comprising the steps of: (a) incubating theindependent sugar moiety B with the biomolecule having sugar moiety A inthe presence of a glycosyltransferase, wherein the sugar moiety A isexcised from the biomolecule and the sugar moiety B is ligated in itsplace, thereby generating the independent sugar moiety A and abiomolecule having sugar B; and (b) isolating the independent sugarmoiety A and the biomolecule having sugar moiety B from step (a),wherein the biomolecule is an enediyne, a vancomycin, a bleomycin, ananthracycline, a macrolide, a pluramycin, an aureolic acid, anindolocarbazole, an aminglycoside, a glycopeptide, a polyene, acoumarin, a benzoisochromanequinone, a calicheamicin, an erythromycin,an avermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.
 2. The method of claim 1, wherein theglycosyltransferase is CalG1, CalG2, CalG3, CalG4, GtfD, GtfE or AveBI.3. The method of claim 1, wherein the biomolecule is an enediyne, avancomycin, a calicheamicin, an erythromycin, an avermectin or anivermectin.
 4. The method of claim 1, wherein the sugar moiety A or B isindependently selected from:


5. The method of claim 1, wherein said exchange is reversible, wherebyincubating the independent sugar moiety A and the biomolecule havingsugar moiety B in the presence of a glycosyltransferase results in theindependent sugar moiety B and the biomolecule having a sugar moiety A.6. A method of generating a biomolecule A, in situ, having a sugarmoiety A from a biomolecule B having the sugar moiety A, the methodcomprising the steps of: (a) incubating the biomolecule A, thebiomolecule B having the sugar moiety A and a nucleotide diphosphate inthe presence of a glycosyltransferase wherein (i) the sugar moiety A ofthe biomolecule B is excised from the biomolecule B, thereby generatingan independent sugar moiety A and a biomolecule aglycon B; and (ii) theindependent sugar moiety A and the biomolecule A are ligated, therebygenerating the biomolecule A having the sugar moiety A; and (b)isolating the biomolecule A having sugar moiety A from step (a), whereinthe biomolecule is an enediyne, a vancomycin, a bleomycin, ananthracycline, a macrolide, a pluramycin, an aureolic acid, anindolocarbazole, an aminglycoside, a glycopeptide, a polyene, acoumarin, a benzoisochromanequinone, a calicheamicin, an erythromycin,an avermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.
 7. The method of claim 6, wherein theglycosyltransferase is CalG1, CalG2, CalG3, CalG4, GtfD, GtfE, EryBV orAveBI.
 8. The method of claim 6, wherein the biomolecule A orbiomolecule B is an enediyne, a vancomycin, a calicheamicin, anerythromycin, an avermectin, an ivermectin or combinations thereof. 9.The method of claim 6, wherein the method of generating biomolecule Ahaving the sugar moiety A from the biomolecule B having the sugar moietyA is reversible, whereby incubating the biomolecule A having the sugarmoiety A and the biomolecule aglycon B in the presence of aglycosyltransferase results in the biomolecule B having the sugar moietyA.
 10. A method of generating a biomolecule A having a sugar moiety Aand a biomolecule B having a sugar moiety B from a biomolecule B havingthe sugar moiety A and a biomolecule A having the sugar moiety Bcomprising the steps of: (a) incubating the biomolecule A having thesugar moiety B, biomolecule B having the sugar moiety A and a nucleotidediphosphate in the presence of a glycosyltransferase wherein (i) thesugar moiety A of the biomolecule B is excised from the biomolecule B,thereby generating an independent sugar moiety A and a biomoleculeaglycon B; (ii) the sugar moiety B of the biomolecule A is excised fromthe biomolecule A, thereby generating an independent sugar moiety B anda biomolecule aglycon A; and (iii) the independent sugar moiety A andthe biomolecule A are ligated and the independent sugar moiety B and thebiomolecule B are ligated, thereby generating the biomolecule A havingthe sugar moiety A and biomolecule B having the sugar moiety B; and (b)isolating the biomolecule A having the sugar moiety A and thebiomolecule B having from the sugar moiety B from step (a)(iii), whereinthe biomolecule is an enediyne, a vancomycin, a bleomycin, ananthracycline, a macrolide, a pluramycin, an aureolic acid, anindolocarbazole, an aminglycoside, a glycopeptide, a polyene, acoumarin, a benzoisochromanequinone, a calicheamicin, an erythromycin,an avermectin, an ivermectin, an angucycline, a cardiac glycoside, asteroid or a flavinoid.
 11. The method of claim 10, wherein theglycosyltransferase is CalG1, CalG2, CalG3, CalG4, GtfD, GtfE, EryBV orAveBI.
 12. The method of claim 10, wherein the biomolecule A orbiomolecule B is an enediyne, a vancomycin, a calicheamicin, anerythromycin, an avermectin, an ivermectin or combinations thereof. 13.The method of claim 10, wherein the method of generating the biomoleculeA having the sugar moiety A and the biomolecule B having the sugarmoiety B is reversible, and whereby incubating the biomolecule A havingthe sugar moiety A and the biomolecule B having the sugar moiety B inthe presence of a glycosyltransferase results in the biomolecule Bhaving the sugar moiety A and the biomolecule A having the sugar moietyB.
 14. A method of generating a library of isolated glycosylatedbiomolecules comprising transferring a sugar moiety from a firstbiomolecule backbone to a second biomolecule backbone in the presence ofa glycosyltransferase wherein the sugar moiety is transferred from thefirst biomolecule backbone to the second biomolecule backbone therebygenerating a non-naturally occurring isolated glycosylated biomolecule,wherein the first or the second biomolecule backbone is an enediyne, avancomycin, a bleomycin, an anthracycline, a macrolide, a pluramycin, anaureolic acid, an indolocarbazole, an aminglycoside, a glycopeptide, apolyene, a coumarin, a benzoisochromanequinone, a calicheamicin, anerythromycin, an avermectin, an ivermectin, an angucycline, a cardiacglycoside, a steroid or a flavinoid.
 15. The method of claim 14, whereinthe first and the second glycosylated biomolecule backbones areindependently selected from: an enediyne, a vancomycin, a calicheamicin,an avermectin, an ivermectin, an erythromycin or combinations thereof.16. The method of claim 14, wherein the sugar moiety is selected from: